Substrate assisted catalysis

ABSTRACT

Novel enzyme mutants are disclosed which are derived from a precursor enzyme by replacing or modifying at least one catalytic functional group of an amino acid residue in a precursor enzyme. Such mutant enzymes have a catalytic preference for substrates which provide the replaced or modified catalytic group or its equivalent such that the substrate together with the enzyme mutant assists in its own catalysis.

This is a division of application Ser. No. 08/090,902 filed Jul. 12,1993, now U.S. Pat. No. 5,371,190 which is a continuation of Ser. No.07/823,039 filed Jan. 14, 1992, now abandoned, which is acontinuation-in part of Ser. No. 07/035,652 filed Apr. 6, 1987, nowabandoned, which is a continuation-in-part of Ser. No. 06/858,594 filedApr. 30, 1986, now abandoned, said Ser. No. 07/823,039 is a continuationof Ser. No. 07/334,081 filed Apr. 4, 1989, now abandoned, which is acontinuation-in-part of Ser. No. 07/127,134 filed Dec. 1, 1987, nowabandoned, which is a continuation-in-part of Ser. No. 06/846,627 filedApr. 1, 1986, now abandoned, which is a continuation-in-part of Ser. No.06/614,615 filed May 29, 1984, now abandoned, and Ser. No. 06/858,594filed Apr. 30, 1986, now abandoned, which is a continuation-in-part ofSer. No. 06/614,612 filed May 29, 1984, now U.S. Pat. No. 4,760,025, andSer. No. 06/614,615 filed May 29, 1984, now abandoned, and Ser. No.06/614,617 filed May 29, 1984, now abandoned, and Ser. No. 06/614,491filed May 29, 1984, now abandoned.

FIELD OF THE INVENTION

The present invention relates to novel enzyme mutants which are derivedfrom a precursor enzyme by replacing or modifying at least one catalyticfunctional group of an amino acid residue in a precursor enzyme. Suchmutant enzymes have a catalytic preference for substrates which providethe replaced or modified catalytic group or its equivalent functionalgroup such that the substrate, in essence, together with the enzymemutant, assists in its own catalytic conversion to product(s)s.

PREFILING DISCLOSURES

Enzymes are polypeptides which catalyze a wide variety of chemicalreactions. It is generally accepted that enzymatic catalysis requiresthat the substrate bind to the enzyme in the region of the enzyme'sactive site such that the specific region being acted upon by the enzymeis distorted into a configuration approximating the transition state ofthe reaction being catalyzed. In many cases the specific site ofcatalysis within the substrate must be oriented so that specificresidues of the enzyme involved in catalysis can act on the bound anddistorted substrate. Thus, within the active site, amino acid residuescan generally be characterized as those primarily involved in substratebinding and hence determinative of substrate specificity and thoseinvolved primarily with the actual chemical catalysis, e.g., thoseinvolved in proton or electron transfer or nucleophilic or electrophilicattack on the substrate.

A wide variety of classical methods have been used to deduce the bindingand catalytic residues in the active site of an enzyme. For example, theX-ray crystal structures of the serine endoprotease subtilisincontaining covalently bound peptide inhibitors (Robertus, J. D., et al.(1972) Biochemistry 11, 2439-2449), product complexes (Robertus, J. D.,et al. (1972) Biochemistry 11, 4293-4303), and transition state analogs(Matthews, D. A., et al. (1975) J. Biol. Chem. 250, 7120-7126; Poulos,T. L., .et al. (1976) J. Biol. Chem. 251, 1097-1103), which have beenreported have provided information regarding the active site ofsubtilisin including the amino acid residues involved in substratebinding and catalytic activity. In addition, a large number of kineticand chemical modification studies have been reported for subtilisinwhich have also aided in deducing the substrate binding and catalyticresidues of subtilisin (Philip, M., et al. (1983) Mol. Cell. Biochem.51, 5-32; Svendsen, I. B. (1976) Carlsberg Res. Comm. 41, 237-291;Markland, F. S. et al. (1971) In The Enzymes Ed. Boyer, P. D., AcademicPress, New York, Vol. 3, pp. 561-608). In most cases where the chemicalmodification was to a catalytic amino acid residue, the enzymaticactivity of the enzymes modified was destroyed or severely impaired(Fersht, A. (1977) "Enzyme Mechanism and Structure", William Freeman,San Francisco, Calif., pp. 201-205). In two reported examples, chemicalmodification of the active site serine of subtilisin resulted in thereplacement of the serine-OH with --SH which produced a modifiedenzymatic activity (Neet, K. E., et al. (1968) J. Bio. Chem. 248,6392-6401; Polgar, L., et al. (1967) Biochemistry 6, 610-620). Mostchemical modifications of catalytic residues, however, necessarilymaintain or increase the effective side chain volume of the amino acidmodified and consequently maintain or decrease the effective volumewithin which the catalytic residues must function.

The recent development of various in vitro techniques to manipulate theDNA sequences encoding naturally-occurring polypeptides as well asrecent developments in the chemical synthesis of relatively shortsequences of single and double stranded DNA has resulted in the reportedsynthesis of various enzymes wherein specific amino acid residues havebeen substituted with different amino acids (Ulmer, K. M. (1983) Science219, 666-671).

There are several reported examples where a catalytic residue of aparticular enzyme has been substituted with a different amino acid. Someof these references describe the replacement of a catalytic amino acidwith an amino acid having a side chain functional group different fromthat of the catalytic amino acid being replaced e.g. substitution of aneutral polar side chain moiety for a side chain moiety containing anacid group or substitution of one nucleophilic side chain moiety with adifferent nucleophilic moiety. Others describe replacements where theside chain functional group of a catalytic residue remained constant butthe position of that functional group was moved within the active site.

For example, Aspartate-102 of eucaryotic trypsinogen is reported to be acatalytic residue required for endoprotease activity. Roczniak, S. O.,et al. (1985), J. Cell Biochem 9B (Abstracts) p. 87 briefly report thesubstitution of asparagine for aspartate at position 102. In this case,the carboxylate of aspartic acid was effectively substituted with thepolar neutral side chain of asparagine which reportedly resulted in adramatic decrease in k_(cat).

Dalbadie-McFarland, G., et al. (1982) Proc. Natl. Acad. Sci. (USA) 79,6409-6413, report the inversion of the ser-thr diad of the β-lactamasegene contained in plasmid pBR322. This inversion resulted in theconversion of the catalytically active Serine-70 to Threonine andreportedly produced a mutant with an ampicillin-sensitive phenotype.

The substitution of Serine-70 in β-lacatamase with cysteine is reportedby Sigal, I. S., et al. (1984) J. Biol. Chem. 259, 5327-5332. Thisreplacement of an active site serine by a cysteine residue results inthe net substitution of an --OH group by an --SH group, each of whichcan be effective nucleophiles. The thiol-containing β-lactamasereportedly catalyses the hydrolysis of β-lactams with a substratespecificity that is distinct from that of the wild type enzyme. Forbenzyl penicillin and ampicillin, the K_(m) values are similar to wildtype values although the k_(cat) values are 1-2% that of a wild typeenzyme. However, when reacted with the cephalosporin nitrocefin, theK_(m) is greater than 10 fold that of the wild type and the k_(cat) isat least as large as the k_(cat) for the wild type enzyme.

In Strauss, et al. (1985) Proc. Natl. Acad. Sci. (USA) 82, 2272-2276,triosphosphate isomerase was reportedly modified to replace glutamicacid at position 165 with aspartic acid. This replacement does not alterthe chemical nature of the side chain at position 165 but rather movesthe catalytic carboxyl group at that position, in essence, by theremoval of a methylene group from glutamic acid. The k_(cat) fordifferent substrates was dramatically altered by this mutation leadingthe author to conclude that glutamic acid at position 165 is criticalfor proton shuttling during catalysis and further suggesting that thisresidue makes only a small contribution to the binding of the reactionintermediates.

The substitution of Serine-102 in the active site of alkalinephosphatase with cysteine is reported by Ghosh, S. S., et al. (1986)Science 231, 154-148. The resulting thiol enzyme catalyzes thehydrolysis of a variety of phosphate monoesters. The authorshypothesize, however, based on the observed catalytic efficiency of thethiol containing enzyme, that the serine to cysteine mutation results ina change in the rate-determining step of catalysis fromdephosphorylation to the formation of a phosphoryl-enzyme intermediate.

The substitution of different amino acids for putative catalyticresidues in various enzymes has been directed to the determination ofwhether these residues are primarily involved in catalysis rather thansubstrate binding. In several reported cases, however, the expectedresult was not obtained. In Gardell, S. J. et al. (1985) Nature 317,551-555, Tyrosine-248 in carboxypeptidase A from rat was substitutedwith phenylalanine. Tyrosine-248 had previously been thought to play arole in catalysis through its phenolic side chain. The particularsubstitution described removed the phenolic hydroxide moiety of tyrosineby substitution with phenylalanine. The authors report that thecatalytic reactivity of the wild type enzyme compared to the substitutedenzyme containing phenylalanine at position 248, for certain substrates,indicated that Tyrosine-248 was not obligatory for the hydrolysis ofpeptide substrates. Rather, the authors suggest that the Tyrosine-248hydroxyl group participates in substrate binding rather than catalysis.

Similarly, Threonine-113 in dihydrofolate reductase from E. coli is astrictly conserved residue at the dihydrofolate binding site whichinteracts with a second conserved residue, Aspartate-27, via a hydrogenbond and presumably with the substrate dihydrofolate indirectly througha water molecule (Jin-Tann Chen, et al. (1985) J. Cell. Biochem 29,73-82). Since Aspartate-27 is also conserved and involved in catalysis,this suggested to the authors that Threonine-113 could be required forproton transfer during catalysis. The authors report the substitution ofThreonine-113 with valine and conclude that Threonine-113 is notinvolved in catalysis since there is no loss of catalytic efficiencyupon substitution with valine.

Schultz, S. C., et al. (1986) Proc. Natl. Acad. Sci. (USA) 83,1588-1592, report the substitution of threonine-71 in class Aβ-lactamase with all possible amino acid substitutions to determine therole of this residue. Threonine-71 is a residue in the conserved triadSer-Thr-Xaa-Lys. The results obtained by these authors suggests thatThreonine-71 is not essential for binding or catalysis, as expected, butis important for stability of the β-lactamase protein.

Much of the work involving the substitution of different amino acids invarious enzymes has been directed to the substitution of amino acidresidues involved in substrate and transition-state binding. Examplesinclude the substitution of single amino acids within the active site oftyrosyl-tRNA synthetase (Cysteine-35→Serine, Winter, G. et al. (1982)Nature 299, 756-758; Cysteine-35→Glycine, Wilkinson, A. J. et al. (1983)Biochemistry 22, 3581-3586; and Threonine-51→Alanine andThreonine-51→Proline, Wilkinson A. J. et al. (1984) Nature 307,187-188).

Other examples of substitutions of amino acids involved in substratebinding include a double mutant of tyrosyl-tRNA synthetase involvingCysteine-35→Glycine together with Threonine-51→Proline (Carter, P. J. etal. (1984) Cell 38, 835-840); the substitution of glycine residues atpositions 216 and 226 of rat pancreatic trypsin with alanine residues toproduce two single substitutions and one double substitution (Craik, C.S. et al. (1985) Science 228, 291-297); and the substitution of variousnon-catalytic residues in dihydrofolate reductase (Villafranca, J. E.,et al. (1983) Science 222, 782-788).

Paluh, J. L., et al. (1984) J. Biol. Chemistry 260, 1188-1894, reportthe substitution of Cysteine-84 with glycine in Serratia marcescensanthranilate synthase Component II. They report that this replacementabolished the glutamine-dependent anthranilate synthase activity but notthe ammonium-dependent activity of the enzyme. They also conclude thatthe mutation provides further evidence for the role of the active siteCysteine-84 in the glutamine amide transfer function of the enzyme. Theauthors also note, however, that the specific amino acid replacementmight cause a relatively minor structural alteration that could abolisha glutamine binding or amide transfer independent of the function ofCysteine-84. It is not clear from this reference whether Cysteine-84 isa residue involved in binding, or actual catalysis.

The substitution of amino acid residue believed to be involved intransition state stabilization of various enzymes have also beenreported. Such work has recently been summarized in Fersht, A. R., etal. (1986), Trends in Biochemical Sciences, 11, 321-325.

In addition to the foregoing, proteases have been used for site-specificproteolysis. Site-specific proteolysis is a powerful and often essentialtool for recovery of heterologous proteins expressed as larger fusionproteins for peptide mapping, and for analysis of structure and foldingby dissection of proteins into functional domains (Jacobsen, H., et al.(1974), Eur. J. Biochem., 45, 623-627) or separate folding units(Richards, F. M., et al. (1959), J. Biol. Chem., 234, 1459-1465).Proteolysis is preferable to chemical cleavage for recovery offunctional proteins because chemical methods have limited specificitiesand usually require extreme conditions that can lead to unwanted sidereactions and product heterogeneity.

Although a number of proteases have been used for site-specificproteolysis (Nagai, K., et al. (1987), Methods Enzymol., 153, 461-481;Craik, C. S., et al. (1985), Science, 228, 291-297; Germino, J., et al.(1984), Proc. Natl. Acad. Sci., 81, 4692-4696; Reinach, F. C., et al.(1986), Nature, 322, 80-83), none appears to be ideally suited. Theutility of these proteases is limited by their substrate specificities(leading to undesirable or incomplete cleavage products) andinstabilities in detergents, reductants, or at high temperatures, whichmay be necessary conditions for solubilizing fusion proteins and makingthe target site accessible for hydrolysis. Furthermore, many of theseproteases, especially mammalian blood-clotting enzymes (Nagai, K., etal. (1987), supra) are unavailable in large quantities and in highlypurified forms so that they are free of other proteolytic activities.Subtilisin BPN' most nearly satisfies all of these requirements for asite-specific protease. Although wild-type subtilisin has been used toobtain specific proteolytic fragments (Jacobsen, H., et al. (1974),supra; Richards, F. M., et al. (1959), supra), its substrate specificityis much too broad to be generally useful.

A reference in another field is Rossman, M. G., et al. (1985) Nature317, 145-153 wherein the RNA of a human rhino virus is postulated to actas a proton acceptor for the autocatalytic cleavage of the viral coatprotein VPO into VP2 and VP4.

The references discussed above are provided solely for their disclosureprior to the filing date of the present case, and nothing herein is tobe construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention or priority basedon earlier filed applications.

Based on the above references, however, it is apparent that thoseskilled in the art have focused on altering enzyme specificity bychanging substrate and transition-state binding residues. It hasheretofore not been recognized that residues containing side chainsdirectly involved in catalysis can be substituted with residuescontaining smaller and catalytically inactive side chains to produceenzyme mutants which are catalytically active with substrates whichprovide the catalytic function of the replaced residue side chain. Thus,these enzyme mutants have a substrate specificity which is distinguishedprimarily at the level of catalysis rather than substrate binding.

Accordingly, it is an object herein to provide enzyme mutants wherein atleast one catalytic group of an amino acid residue of a precursor enzymeis replaced or modified such that the thus formed mutant enzyme has apreferred catalytic activity for a substrate which is capable ofproviding the replaced or modified catalytic function when in contactwith the mutant enzyme.

It is a further object to provide DNA sequences encoding such enzymemutants as well as expression vectors containing such mutant DNAsequences.

Still further, another object of the present invention is to providehost cells transformed with such vectors as well as host cells which arecapable of expressing such enzyme mutants either intracellularly orextracellularly.

A further object of the present invention is to provide a catalyticallyactive mutant enzyme substrate complex wherein at least one of thecatalytic functional group, of the complex is provided by the substrate.

Still further, an object of the present invention is to provideprocesses wherein the enzyme mutants of the invention are contacted withmodified substrates to bring about desired enzymatic catalysis.

Further, it is an object of the invention to provide fusion polypeptidecontaining a target sequence which is reactive with the enzyme mutantsof the invention.

Still further, it is an object herein to provide solid supportscontaining the enzyme mutants of the invention as well as methodsutilizing such solid support.

A further object is to provide methods for purifying the enzyme mutantsof the invention.

SUMMARY OF THE INVENTION

The invention includes enzyme mutants not found in nature which arederived from a precursor enzyme by the replacement or modification of atleast one catalytic group of an amino acid residue which when in contactwith a selected region of a polypeptide substrate functionscatalytically therewith. The enzyme mutant so formed is relativelyinactive catalytically with the corresponding substrate as compared tothe mutant's catalytic activity with a modified substrate formed byreplacing or modifying a moiety in a selected region of the precursorenzyme's substrate. This selected region of the substrate is modified toinclude the catalytic group, or its equivalent which is replaced ormodified in the precursor enzyme, such that the enzyme mutant iscatalytically active with the modified substrate.

The invention also includes mutant DNA sequences encoding such mutantenzymes, expression vectors containing such mutant DNA sequences andhost cells transformed with such vectors which are capable of expressingsaid enzyme mutants.

The invention also includes a catalytically active enzyme-substratecomplex comprising an enzyme mutant and a modified substrate. The enzymemutant is not found in nature and is derived from a precursor enzyme bythe replacement or modification of at least one catalytic group of anamino acid residue which, when in contact with a selected region of asubstrate for the precursor enzyme, functions catalytically with suchsubstrate. The enzyme mutant so formed is relatively inactive with thesubstrate for the precursor enzyme as compared to the enzyme mutant'scatalytic activity with a modified substrate. The modified substrate isformed by replacing or modifying a moiety in the selected region of theprecursor enzyme's substrate. This selected region of the substrate ismodified to include the catalytic group, or its equivalent, which isreplaced or modified in the precursor enzyme such that the enzyme mutantis catalytically active with the modified substrate.

The invention further includes a process comprising contacting an enzymemutant and a modified substrate to produce substrate assisted catalysisof the modified substrate. In this aspect of the invention, the enzymemutant is the same as that defined for the enzyme mutant-substratecomplex of the invention.

The invention also includes fusion polypeptides comprising an aminoterminal portion, a carboxy terminal portion and a target cleavagesequence between the amino and carboxy terminal portions. The targetcleavage sequences comprises amino acid residues P₄, P₃, P₂ and P₁ whereP₂ is histidine. This target cleavage site is reactive with H64A mutantsubtilisins.

The invention further includes subtilisin mutants comprising the aminoacid sequence of B. amyloliquefaciens subtilisin or equivalents thereofwherein the mutant amino acid sequence contains the substitution H64Aalone or in combination with substitutions at other residues.

Further, the invention includes processes for cleaving the above fusionpolypeptides with the above H64A mutant subtilisins.

Further, the invention includes processes for purifying H64A subtilisinmutants wherein at least one surface amino acid residue of a H64Asubtilisin mutant is substituted to contain cysteine. Thecysteine-containing mutant is thereafter contacted with chromatographicmaterial having an affinity for cysteine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are the DNA and amino acid sequence for B. amylolquefacienssubtilisin.

FIG. 2 depicts catalytic residues of B. amylolquefaciens subtilisin.

FIGS. 3A and 3B depict the amino acid sequence of subtilisin as obtainedfrom various sources.

FIG. 3C depicts the conserved residues of B. amylolquefaciens subtilisinwhen compared to a number of other subtilisin sequences.

FIG. 4 is a schematic diagram showing the substrate binding cleft tosubtilisin together with a substrate.

FIG. 5 is a stereo view of B. amylolquefaciens subtilisin containing amodeled bound peptide substrate having the sequenceL-Phe-L-Ala-L-His-L-Tyr-L-Gly-L-Phe representing residues P4 to P2' ofthe substrate.

FIG. 6 depicts the plasmid PS4.

FIG. 7A and 7B depict the pH dependence of hydrolysis ofN-succinyl-L-Phe-L-Ala-L-Ala-L-Phe-ρ-nitroanilide substrate (sFAAF-pna)by S24C:H64A subtilisin BPN'.

FIG. 7C depicts the pH dependence for hydrolysis ofN-succinyl-L-Phe-L-Ala-L-His-L-Phe-ρ-nitroanilide substrate (sFAHF-pna)by S24C:H64A subtilisin BPN'.

FIGS. 8A and 8B depict the hydrolysis of a polypeptide substrate byS24C:H64A and S24C subtilisin BPN'.

FIG. 9 depicts a stereo view of a complex between bovine trypsin andpancreatic trypsin inhibitor complex.

FIG. 10 is a stereo view of a substrate model (filled atoms)L-Phe-L-Ala-L-His-L-Tyr-L-Ala-L-Phe bound to the active site of Bacillusamyloliquefaciens subtilisin BPN' (open atoms). This model shows thesuperposition of the catalytic histidine (H64) with the substrate P2histidine. The substrate may be represented as: ##STR1## where thescissile peptide bond is between the P1 and P1' residues (Schechter, I.,et al. (1967), Biochem. Biophys. Res. Commun., 27, 157-162).

FIG. 11 depicts the activity of S24C:H64A subtilisin BPN' with sFAHF-pnain the presence of (A) salts: KCl (O), NaCl (•); (B) denaturants: urea(□), guanidine hydrochloride (GuHCl, ), (C) anionic detergents: SDS (•),sodium deoxycholate (□); (D) nonionic detergents: nonidet P-40 (O),tween 20 ( ).

FIG. 12 depicts the construction of phagemid pZAP encoding a fusionprotein for the signal (S) and one synthetic domain for Staphylococcusaureus protein A (Z) followed by a histidine-containing linker (L) andthen E. coli alkaline phosphatase (AP). The residues in the target siteto be cleaved by the engineered subtilisin BPN' variant are designatedP4 through to P2' (scissile bond indicated by large arrow).

FIG. 13 depicts digestion of the Z-AP fusion protein with mutantsubtilisins. 200 pmol Z-AP was incubated without enzyme or with 10 pmolof either S24C:H64A or S24C:H64A:E156S:G169A:Y217L subtilisin BPN'variants in 100 μl 1 100 mM Tris-HCl at pH 8.60, 1 mM PMSF, 0.1% (v/v)tween 20 in the presence (+) or absence (-) or 2M KCl at 37° C. for thetimes indicated (hr). Samples were analyzed by SDS-PAGE. Molecularweight standards (M_(r)) have sizes in kilodaltons as indicated.

FIG. 14 depicts the combination of the plasmid encoding fusion proteinZ-bIGF-I, showing details of the target cleavage sequence and thejunction with the protein A Z domain and b-IGF-I.

FIG. 15 shows the mass spectrum obtained from bIGF-I isolated fromZ-bIGF-I after cleavage with S24C:H64A: E156S:G169A:Y217L subtilisinBPN'.

FIG. 16 is a stereo view of subtilisin BPN' showing the location of thecatalytic triad (Ser221, His64 and Asp32) in relation to residue 24which was used for immobilization of the enzyme after mutating it tocysteine (S24C). The distance between CA His64 and CA Ser24 is 24 Å.Additional residues (Thr22 and Ser87) are also shown.

FIG. 17 demonstrates the effect of imidazole on thenon-histidine-containing substrate sFAAF-pna by S24C:H64A subtilisinBPN'.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that a catalytic group in an amino acidside chain in an enzyme can be replaced or modified to produce a mutantenzyme which is reactive with substrates which contain the replaced ormodified catalytic group. The replaced or modified catalytic group islocated in the substrate such that it is able to assist, with the mutantenzyme, in the catalysis of the modified substrate.

Specifically, B. amyloliquefaciens subtilisin, an alkaline bacterialprotease, has been mutated by modifying the DNA encoding subtilisin toencode the substitution of the catalytic residue His-64 with alanine. Asexpected, k_(cat) and the catalytic efficiency, as measured by k_(cat)/K_(m), of this mutant enzyme was significantly reduced as compared tothe wild type subtilisin when contacted with substrates readily cleavedby wild type subtilisin. Surprisingly, various substrates containinghistidine in the P2 position were preferred by the Ala-64 mutantsubtilisin.

Previous studies have focused on altering enzyme specificity by changingresidues on the enzyme that bind the substrate. The alternative approachdescribed herein, termed "substrate-assisted catalysis", is applicableto a wide range of enzymes and substrates other than those specificallydisclosed herein. In general, the invention is applicable to any enzymein which part of the enzyme is removed and appropriately supplied by asimilar functionality from a bound substrate. In this way substrates aredistinguished primarily at the level of catalysis instead of binding,permitting the design of extremely specific enzyme mutants.

Since, in the case of proteases, such enzyme mutants have a specificityfor substrates having the catalytic functionality which the mutantenzyme lacks, it is possible to design fusion polypeptides containing atarget cleavage sequence which provides a similar functionality. In sodoing, the reactivity of the fusion polypeptide with the mutantproteases of the invention is significantly restricted to the targetcleavage sequence.

As used herein, "enzymes" are polypeptides which either alone or inconjunction with various co-factors catalyze a covalent change in asubstrate. Enzymes can be categorized according to a systematicnomenclature and classification which has been adopted on therecommendation of the International Enzyme Commission. Thus, enzymes canbe categorized as oxidoreductases (enzymes involved in oxidationreduction reactions), transferases (enzymes involved in the transfer offunctional groups), hydrolases (enzymes involved in hydrolyticreactions), lyases (enzymes catalyzing addition reactions to doublebonds), isomerases (enzymes involved in isomerization reactions) andligases (enzymes involved in the formation of bonds with ATP cleavage).See, generally, Lehninger, A. L., Biochemistry, Worth Publishers, Inc.,New York, N.Y. (1970), pp. 147-187.

A "precursor enzyme" refers to an enzyme in which a catalytic amino acidresidue can be replaced or modified to produce a mutant enzyme.Typically, the DNA sequence encoding the precursor enzyme may bemodified to produce a mutant DNA sequence which encodes the substitutionof one or more catalytic amino acids in the precursor enzyme amino acidsequence. Suitable modification methods are disclosed herein and in EPOPublication No. 0130756 published Jan. 9, 1985. The precursor enzyme,however, can also be modified by means other than recombinant DNAtechnology to produce the mutant enzyme of the invention.

A precursor enzyme may also be a recombinant enzyme which refers to anenzyme for which its DNA has been cloned or to an enzyme in which thecloned DNA sequence encoding an enzyme is modified to produce arecombinant DNA sequence which encodes the substitution, deletion orinsertion of one or more amino acids in the sequence of a naturallyoccurring enzyme. Suitable methods to produce such modifications includethose disclosed herein and in EPO Publication No. 0130756. For example,the subtilisin multiple mutant herein containing the substitution ofserine at amino acid residue 24 with cysteine and the substitution ofhistidine at amino and residue 64 with alanine can be considered to bederived from the recombinant subtilisin containing the substitution ofcysteine for serine at residue 24. The mutant thus is produced by thesubstitution of alanine for histidine at residue 64 in the Cys-24recombinant subtilisin. The resulting double mutant is designatedS24C:H64A where the single letter code (Creighton, Thomas E. (1984)Proteins, Structures and Molecular Properties, International StudentEdition, W. H. Freeman and Company, p. 7) for the wild-type amino acidis followed by residue number and the amino acid replacement.

Other examples of recombinant subtilisin which have been modified tosubstitute alanine for histidine at residue 64 include the following:S24C:G166A, S24C:E156S:G169A:Y217L and S24C:E156S:G166A:G169A: Y217L.These particular recombinant subtilisin mutants were combined with theH64A mutation to increase the catalytic activity of such mutants withsubstrates providing the missing histidine functionality in the mutantsubtilisin.

Carbonyl hydrolases are enzymes which hydrolyze compounds containing##STR2## bonds in which X is oxygen, nitrogen or sulfur. They includenaturally-occurring carbonyl hydrolases and recombinant or chemicallysynthesized carbonyl hydrolases. Naturally occurring carbonyl hydrolasesprincipally include hydrolases, e.g. lipases and peptide hydrolases,e.g. subtilisins or metalloproteases. Peptide hydrolases includeα-aminoacylpeptide hydrolase, peptidylamino-acid hydrolase, acylaminohydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiolproteinase, carboxylproteinase and metalloproteinase. Serine, metallo,thiol and acid proteases are included, as well as endo andexo-proteases.

Subtilisins are bacterial carbonyl hydrolases which generally act tocleave peptide bonds of proteins or peptides. As used herein,"subtilisin" means a naturally occurring subtilisin or a recombinantsubtilisin. A series of naturally occurring subtilisins is known to beproduced and often secreted by various bacterial species. Amino acidsequences of the members of this series are not entirely homologous.However, the subtilisins in this series exhibit the same or similar typeof proteolytic activity. This class of serine proteases shares a commonamino acid sequence defining a catalytic triad which distinguishes themfrom the chymotrypsin related class of serine proteases. The subtilisinsand chymotrypsin related serine proteases both have a catalytic triadcomprising aspartate, histidine and serine. In the subtilisin relatedproteases the relative order of these amino acids, reading from theamino to carboxy terminus is aspartate-histidine-serine. In thechymotrypsin related proteases the relative order, however, ishistidine-aspartate-serine. Thus, subtilisin herein refers to a serineprotease having the catalytic triad of subtilisin related proteases.

Carbonyl hydrolases and their genes may be obtained from manyprocaryotic and eucaryotic organisms. Suitable examples of procaryoticorganisms include gram negative organisms such as E. coli or pseudomonasand gram positive bacteria such as micrococcus or bacillus. Examples ofeucaryotic organisms from which carbonyl hydrolase and their genes maybe obtained include yeast such as S. cerevisiae, fungi such asAspergillus sp., and mammalian sources such as, for example, Bovine sp.from which the gene encoding the carbonyl hydrolase chymosin can beobtained. As with subtilisins, a series of carbonyl hydrolases can beobtained from various related species which have amino acid sequenceswhich are not entirely homologous between the members of that series butwhich nevertheless exhibit the same or similar type of biologicalactivity. Thus, carbonyl hydrolase as used herein has a functionaldefinition which refers to carbonyl hydrolases which are associated,directly or indirectly, with procaryotic and eucaryotic sources.

An "enzyme mutant" has an amino acid sequence which is derived from theamino acid sequence of a "precursor enzyme" and has a catalyticpreference for a modified substrate or a target cleavage sequence asdefined herein. The amino acid sequence of the enzyme mutant may be"derived" from the precursor amino acid sequence by the substitution ofone or more catalytic amino acid residues of the precursor amino acidsequence. Suitable methods for such manipulation of the precursor DNAsequence include methods disclosed herein and in EPO Publication No.0130756. Other methods, for example, to directly modify the amino acidside chain of the precursor enzyme may be used provided they produce thecatalytic preference for a modified substrate or a target cleavagesequence. A "catalytic amino acid residue" is one which contains acatalytic group.

As used herein in connection with enzyme mutants, a "catalytic group" inan enzyme is a functional side chain of an amino acid residue whichundergoes a change in charge or chemical bonding state during a reactionsequence and which becomes regenerated at the end of the reactionsequence, or which interacts directly with such a functional side chainto facilitate its change in charge or chemical bonding state. Catalyticgroups typically participate in catalysis by interacting directly orindirectly as a nucleophile, electrophile, acid, base or electrontransfer agent with the reactive site of a substrate. Typical catalyticamino acid residues and their respective catalytic groups (shown inparentheses) include: Ser(--OH), Thr(--OH), Cys(--SH), Tyr(--OH),Lys(--NH₂), Asp(--CO₂ H), Glu(--CO₂ H), His(imidazolyl) and Met(--SCH₃).See Table I. Thus, for example, catalytic groups for B.amyloliquefaciens subtilisin and as shown in FIG. 2 corresponding to theamino acid position numbers referred to in FIG. 1 comprise the sidechains to the amino acids Asp-32, His-64 and Ser-221.

                  TABLE I                                                         ______________________________________                                        Precursor Enzyme                                                                     Preferred                                                                     Amino     Alternate  Modified Substrate                                Catalytic                                                                            Acid      Amino Acid         Equivalent                                Catalytic                                                                            Residue   Residue    Catalytic                                                                             Catalytic                                 Residue                                                                              Substitution                                                                            Substitution                                                                             Group   Group                                     ______________________________________                                        His    Gly, Ala  Ser        Imidazoyl                                                                             --NH.sub.2                                Lys    Gly, Ala, Thr, Leu,  --NH.sub.2                                                                            Imidazolium                                      Ser       Asn                                                          Ser    Gly       Ala        --OH    --SH                                      Thr    Gly       Ala        --OH    --SH                                      Cys    Gly       Ala        --SH    --OH                                      Asp    Gly, Ala  Ser        --CO.sub.2 H                                                                          Imadazoyl,                                                                    Phenol                                    Glu    Gly, Ala  Thr, Cys   --CO.sub.2 H                                                                          Imadazoyl,                                       Ser                          Phenol                                    Tyr    Gly, Ala, Thr, Cys   Phenol  --OH, --SH                                       Ser, Asn,                    Imidazolium                                      Gln                                                                    Met    Gly, Ala  Ser, Thr   --S--CH.sub.3                                                                         --SH                                      Phe    Gly, Ala, Leu, Val   Phenyl  --S--CH.sub.3,                                   Ser                          Phenol                                    Trp    Gly, Ala, Leu, Val   Indole  --S--CH.sub.3,                                   Ser                          Phenol,                                                                       Phenyl                                    ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Precursor Enzyme                                                                     Preferred                                                                     Amino     Alternate  Modified Substrate                                Catalytic                                                                            Acid      Amino Acid         Equivalent                                Catalytic                                                                            Residue   Residue    Catalytic                                                                             Catalytic                                 Residue                                                                              Substitution                                                                            Substitution                                                                             Group   Group                                     ______________________________________                                        Asn    Gly, Ala, Thr, Ser, Cys                                                                             ##STR3##                                                                              ##STR4##                                 Gln    Gly, Ala, Thr, Ser, Cys                                                                             ##STR5##                                                                              ##STR6##                                 Arg    Gly, Ala, Thr, Ser, Cys                                                                             ##STR7##                                                                              ##STR8##                                 ______________________________________                                    

As used herein in connection with the enzyme-substrate complexes orprocesses of the invention, a "catalytic group" in addition to the abovedefinition, includes functional side chains of amino acid residues whichaid in stabilizing the transition state of a reaction by interactingdirectly or indirectly with a polarized or charged transition state.Such transition state stabilization is typically achieved by theformation of salt bridges or the creation of a dipole-dipole interaction(e.g. hydrogen bond formation) between the transition state and thecatalytic residues stabilizing it. Typical catalytic amino acid residuesinvolved in transition state stabilization and their respectivecatalytic groups (shown in parenthesis) include those catalytic residuesof Table I: ##STR9## (See Table II). For the B. amyloliquefacienssubtilisin shown in FIGS. 1 and 2, a catalytic residue involved intransition state stabilization is Asn-155 which provides a hydrogen bondto stabilize the oxyanion of the tetrahydral intermediate shown in FIG.2.

Many enzymes are sufficiently characterized such that the catalyticgroups of these enzymes (as defined above) are well known to thoseskilled in the art. However, for those enzymes which are not socharacterized, the catalytic residues can be readily determined.

In this regard, amino acid replacement or chemical modification ofcatalytic groups (including those directly involved in catalysis andthose involved in transition state stabilization) typically cause largedisruptions in the catalytic step of the reaction (e.g. often measuredby k_(cat)) and little effect on the enzyme substrate dissociationconstant (e.g. often measured by K_(m)).

Thus, to determine whether a putative catalytic group is indeedcatalytic, one skilled in the art can replace or modify the residuecontaining that group as described herein. If such substitution ormodification abolishes or significantly reduces k_(cat), but does notsubstantially effect K_(m) (e.g. increases or decreases K_(m) by afactor of 50 or preferably 10 or less), the side chain of the residuesubstituted is a catalytic group.

Structural methods such as x-ray crystallography and nuclear magneticresonance spectroscopy (nmr) can also be used to identify potentialcatalytic groups by their proximity to the site of the substratechemical bond which becomes altered. Chemical, kinetic and nmr methodscan also be useful in identifying catalytic groups by showing a changein their charge or chemical bonding properties during a reaction.

Alternatively, if a particular enzyme is not well characterized but isclosely related to an enzyme wherein one or more catalytic groups arealready well-defined, the catalytic groups in that enzyme may beidentified by determining its equivalent catalytic residues.

Thus, for example, a catalytic residue (amino acid) of a precursorcarbonyl hydrolase is equivalent to a residue of B. amyloliquefacienssubtilisin if it is either homologous (i.e., corresponding in positionin either primary or tertiary structure) or analogous to a specificresidue or portion of that residue in B. amyloliquefaciens subtilisin(i.e., having the same or similar functional capacity to combine, react,or interact chemically).

In order to establish homology to primary structure in the aboveexample, the amino acid sequence of a precursor carbonyl hydrolase isdirectly compared to the B. amyloliquefaciens subtilisin primarysequence and particularly to a set of residues known to be invariant inall subtilisins for which sequence is known (FIG. 3C). After aligningthe conserved residues, allowing for necessary insertions and deletionsin order to maintain alignment (i.e., avoiding the elimination ofconserved residues through arbitrary deletion and insertion), theresidues equivalent to the catalytic amino acids (His-64, Asp-32,Ser-221, Asn-155) in the primary sequence of B. amyloliquefacienssubtilisin are defined. Alignment of conserved residues preferablyshould conserve 100% of such residues. However, alignment of greaterthan 75% or as little as 20% of conserved residues is also adequate todefine equivalent residues.

For example, in FIG. 3A the amino acid sequence of subtilisin from B.amyloliquefaciens (BPN') B. subtilisin var. I168 and B. lichenformis(Carlsbergensis) are aligned to provide the maximum amount of homologybetween amino acid sequences. A comparison of these sequences shows thatthere are a number of conserved residues contained in each sequence.These residues are identified in FIG. 3C.

These conserved residues thus may be used to define the correspondingequivalent catalytic amino acid residues of B. amyloliquefacienssubtilisin in other carbonyl hydrolases such as thermitase derived fromThermoactinomyces. These two particular sequences are aligned in FIG. 3Bto produce the maximum homology of conserved residues. As can be seenthere are a number of insertions and deletions in the thermitasesequence as compared to B. amyloliquefaciens subtilisin. Thus, theequivalent catalytic amino acid of Asn-155 in B. amyloliquefacienssubtilisin in thermitase is the particular asparagine shown beneathAsn-155.

Equivalent catalytic residues homologous at the level of tertiarystructure for a precursor carbonyl hydrolase whose tertiary structurehas been determined by x-ray crystallography, are defined as those forwhich the atomic coordinates of 2 or more of the main chain atoms of aparticular amino acid residue of the precursor carbonyl hydrolase and B.amyloliquefaciens subtilisin (N on N, CA on CA, C on C, and O on O) arewithin 0.13 nm and preferably 0.1 nm after alignment. Alignment isachieved after the best model has been oriented and positioned to givethe maximum overlap of atomic coordinates of non-hydrogen protein atomsof the carbonyl hydrolase in question to the B. amyloliquefacienssubtilisin. The best model is the crystallographic model giving thelowest R factor for experimental diffraction data at the highestresolution available. ##EQU1##

Equivalent catalytic residues which are functionally analogous to acatalytic residue of B. amyloliquefaciens subtilisin are defined asthose amino acids of the precursor carbonyl hydrolases which may adopt aconformation such that they either alter, modify or contribute tocatalysis in a manner defined and attributed to a specific catalyticresidue of the B. amyloliquefaciens subtilisin as described herein.Further, they are those residues of the precursor carbonyl hydrolase(for which a tertiary structure has been obtained by x-raycrystallography), which occupy an analogous position to the extent thatalthough the main chain atoms of the given residue may not satisfy thecriteria of equivalence on the basis of occupying a homologous position,the atomic coordinates of at least two of the side chain atoms of theresidue lie with 0.13 nm of the corresponding side chain atoms of acatalytic group of B. amyoliquefaciens subtilisin. The three dimensionalstructures would be aligned as outlined above.

As indicated, amino acid residues of B. amyloliquefaciens subtilisinhave been modified by substitution with a different amino acid toincrease the catalytic activity of H64A mutants, e.g., at residues G166,E156, G169, Y217. Equivalent residues in other carbonyl hydrolases maybe similarly identified as homologous to the primary sequence or to thetertiary structure of B. amyloliquefaciens subtilisin. Further,equivalent residues may be identified as functionally analogous to aparticular residue of B. amyloliquefaciens subtilisin. In this regardspecific reference is made to EPO Publication No. 0251446.

As used herein, a "substrate" refers to a substrate which is reactivewith a precursor enzyme. For those enzymes which utilize polypeptides assubstrate, the substrate is typically defined by an amino acid sequencewhich is recognized by the precursor enzyme to bind the substratetherewith. For example, a substrate for trypsin contains the amino acidsequence

    X.sub.1 --K--X.sub.2 or X.sub.1 --R--X.sub.2

where X₁ is any P₂ amino acid, X₂ is a P₁ ' amino acid except Pro, R isArg and K is Lys. Subtilisin is broadly specific and will cleave a widerange of polypeptide substrates albeit at substantially different rates.An example of a substrate which is efficiently cleaved is the sequenceFAAY↓AF (at the point designated by the arrow) where F, A, Y are Phe,Ala and Tyr, respectively, and Tyr occupies the P₁ position (see FIG.4). These residues, in general, are those recognized by the enzyme tobind a particular substrate and are referred to herein as a "selectedregion" of the substrate. Of course, there may be a wide range ofpolypeptides which are substrates for a particular precursor enzyme.However, each of these substrates will contain a selected regionrecognized by the precursor enzyme.

In various aspects of the invention, the substrate may be anon-proteinatious molecule such as a nucleic acid, carbohydrate, ametabolite in a biological pathway or an antibiotic. In the case ofnucleic acids and carbohydrates the "substrate" can be similarly definedas for a polypeptide substrate except that these regions are defined notby amino acid sequence but rather by nucleic acid sequence andcarbohydrate sequence, respectively. Thus, for example, restrictionendonucleases recognize specific DNA sequences and α amylase recognizesthe α(1,4) glycosidic linkage between glucose molecules in amylose.

In the case of antibiotics, which usually are neither polypeptides,nucleic acids or carbohydrates, there is usually little problem inidentifying the substrate. Thus, for example, the substrates for βlactamases are penicillins and cephalosporans.

In general, substrates for a wide range of enzymes, including theselected region of such substrates, are known to or may be readilydetermined by those skilled in the art.

A "modified substrate" is a substrate wherein at least one moietycontained therein is replaced or modified to form a modified moietywhich includes the catalytic group replaced or modified in a precursorenzyme or the equivalent of the catalytic group so replaced or modified.The catalytic group contained in the modified substrate is positionedsuch that upon binding with the mutant enzyme formed by modifying aparticular precursor enzyme, the modified substrate provides a modifiedmoiety which when in contact with the mutant enzyme provides thecatalytic group replaced in the precursor enzyme or its equivalent. Thethus formed enzyme mutant-modified substrate complex is thereby renderedcatalytically active.

In the case of precursor enzymes having corresponding polypeptidesubstrates, the modified substrate is also a polypeptide which containsan amino acid sequence which binds to the mutant enzyme and whichcontains an amino acid residue within that selected region which has aside chain catalytic group which is the same or equivalent to the aminoacid replaced or modified to form the mutant enzyme. This amino acidsequence while binds to and is reactive with the mutant enzyme issometimes also referred to as the "target cleavage sequence".

In a preferred embodiment of the invention, a family of H64A subtilisinBPN' mutants (from B. amylolquefaciens) are disclosed. Such H64A mutantshave a specificity for target cleavage sequences designated P₄, P₃, P₂and P₁. This designation corresponds to the analogous designation foramino acid residues in the substrate for wild type subtilisin (see FIG.4). For the H64A mutant subtilisin, P₂ in the target cleavage sequenceis histidine. Further, this target cleavage sequence preferably containsP₁ amino acids comprising tyrosine, phenylalanine, methionine, leucine,and lysine. Most preferably, P₁ is tyrosine or phenylalanine. The P₄residue in this target cleavage sequence is preferably phenylalanine,isoleucine, methionine, alanine, leucine, lysine or valine. Mostpreferably P₄ is phenylalanine. The P₃ residue in the target cleavagesequence may be any amino acid but is preferably alanine, asparagine,glutamate, aspartate, isoleucine, glutamine, tyrosine, histidine,glycine, leucine, serine or valine. Most preferably P₃ is alanine,valine, glutamine or histidine. In many instances, the residues P₁ andP₂ are sufficient to define the target cleavage sequence such that amutant H64A subtilisin will cleave at the C terminal side of the P₁residue. However, the preferred target cleavage sequence comprises P₄through P₁ and most preferably comprise the sequencesphenylalanine-alanine-histidine-tyrosine andphenylalanine-alanine-histidine-phenylalanine.

An "equivalent" catalytic group in a modified substrate is one which iscapable of reacting, combining or interacting in the same or similarmanner as that which was removed from or modified in the precursorenzyme. Equivalent catalytic group refers to a group having the abilityto provide a similar or equivalent catalytic role. It is not necessarythat an equivalent catalytic group provide equivalent chemicalstructure. For example, if a catalytic His residue is removed from theenzyme, an equivalent catalytic group from the substrate would be animidazolyl group which may be donated (but not always or exclusively) bya His side chain. If a catalytic Ser residue is removed from the enzymean equivalent catalytic group from the substrate may be a hydroxyl groupwhich may be donated by threonine or tyrosine side chain. In some casesthe equivalent catalytic group may not be identical to the originalenzyme group. Thus, an equivalent catalytic group for the Serine--OH maybe the Cysteine--SH. See Tables I and II.

Upon binding with the enzyme mutant, the same or equivalent catalyticgroup in the modified substrate is capable of being positioned such thatit is close to the original position of the side chain of the amino acidresidue substituted or modified in the precursor enzyme. In this mannerthe catalytic function of the enzyme mutant can be greatly enhanced whenthe enzyme mutant binds a modified substrate.

The positioning of the catalytic group or equivalent catalytic groupwithin the selected region of a substrate to form a modified substratemay be achieved by substituting each of the amino acid residues withinthe selected region with a different amino acid to incorporate thecatalytic or equivalent group in the modified substrate at variouspositions. Such modified substrates can be readily made by the methodsdisclosed herein and by methods known to those skilled in the art.Thereafter, these modified substrates are contacted with the particularmutant enzyme to determine which, if any, of the modified substrates arereactive with the enzyme mutant.

Alternatively, if the crystal structure of a particular enzyme or enzymesubstrate complex is known, model building may be utilized to determinehow a modified substrate should be constructed. Such model building mayuse, for example, a FRODO program (Jones, T. A. (1978) J. Appl.Crystallogr. 11, 268) in conjunction with an Evans and Sutherland PS300graphics system. For example, one can use such a program and graphicsystem to construct the stereo view of B. amylolquefaciens subtilisinshown in FIG. 5 containing a model bound peptide substrate having thesequence L-Phe-L-Ala-L-His-L-Tyr-L-Gly-L-Phe representing residuesP4-P2' of the substrate.

A two-dimensional representation of the relationship between thesubsites involved in subtilisin (S4 through S3') and the substrateresidues involved in a substrate binding (P4 through P3') is shown inFIG. 4. Normally, the P2 position in the substrates that are typicallyreactive with subtilisin do not require histidine.

The model in FIG. 5 is based upon a 2.0 Å X-ray crystallographic studyof product complexes bound to subtilisin. See e.g. Robertus, J. D. etal. (1972) Biochemistry 11, 4293; Poulos, T. L. et al. (1976) J. Biol.Chem. 251, 1097. The catalytic triad (Asp-32, His-64, and Ser-221) isshown with the His P2 side chain from the substrate superimposed uponthe catalytic His-64. The distances between the Oγ of Ser-221 and thecorresponding NεZ nitrogens from His-64 and the modeled P2 His sidechain are 3.17 Å and 3.17 Å, respectively. The distances between the OδZof Asp-32 and the corresponding Nδ1 nitrogens from His-64 and themodeled P2 His side chain are 2.72 Å and 2.72 Å, respectively. Themodeled distances between the NεZ and Nδ1 nitrogens of the histidinesare 1.39 Å and 1.35 Å, respectively. The hydrogen bond distances anddihedral angles for the stereo view of the complex of FIG. 4 are givenin Table III as subtilisin model S1.

Likewise, one can generate a stereo view of a complex between bovinetrypsin and pancreatic trypsin inhibitor (PTI) complex in which theequivalent P2 substrate side chain (Cys-14 in PTI) is replaced by Hisand superimposed upon His-57 in trypsin. The coordinates for thetrypsin/trypsin inhibitor complex were taken from the Brookhaven ProteinData Bank entry 2PTC deposited by R. Huber and J. Deisenhofer, 9/82. Seealso Deisenhofer, J., et al. (1975) Acta. Crystallogr., Sect. B., 31,238. The catalytic triad of trypsin (Ser-195, His-57, Asp-102) is shownand the carbonyl carbon of Lys-15 at the P1 position in PTI is labeled.The hydrogen bond distances and dihedral angles for this stereo view inFIG. 9 are given as trypsin model T1 in Table III.

TABLE III

Pertinent bond angles and distances modeled for substrate-assistedcatalysis by a His P2 side chain in subtilisin or trypsin as depicted inFIGS. 5 and 9, respectively. Dihedral angles for the His side chains aredefined by χ¹ (N-Cα-Cβ-Cγ) and χ² (Cα-Cβ-Cγ-Cδ) . The hydrogen bondangles (Nε2(His)-Hδ(ser)-Oγ(Ser)) were calculated from the measuredCβ(Ser)-Oγ(Ser)-Nε2(His) angle, the Nε2(His)-Oγ(Ser) bond distance andthe known Oγ(Ser)-Hδ(Ser) distance (0.96 Å) and theCβ(Ser)-Oγ(Ser)-Hδ(Ser) bond angle (108.5°) (Weiner, S. J. et al. (1984)J. Am. Chem. Soc. 106., 765). H-bond distances were measured between thecatalytic Ser(O) and Asp (Oδ1 and Oδ2) to the Nε2 and Nδ1, respectively,from the enzyme His or the substrate His P2. The distances are givenbetween the enzyme His and the modeled substrate His P2 Nε2 and Nδ1nitrogens. Model 1 (shown in FIG. 5 and 9 for subtilisin and trypsin,respectively) has the His P2 side chain optimized for H-bond distancesbetween the imidazoyl nitrogen, Nε2 and Nδ1, to the catalytic Ser andAsp, respectively. Model 2 (graphic view not shown) has idealized anglesfor the His P2 side chain.

                                      TABLE III                                   __________________________________________________________________________            Angles       Distance (Å)                                                 Dihedral                                                                             H-bond                                                                              Nε2 (his)→                                                           Nδ1 (His)→                                                                   Catalytic                                                                          His→His P2                         x.sup.1                                                                           x.sup.2                                                                          (Ser→His)                                                                    Og(Ser)                                                                             Oδ1(Asp) or Oδ2(Asp)                                                          Nε2/Nε2                                                            Nδ1/Nδ1               __________________________________________________________________________    Subtilisin                                                                    Catalytic His64                                                                       -167°                                                                       85°                                                                      148°                                                                         3.17  3.34  2.27  --   --                                (actual)                                                                      His P2 side                                                                   chain                                                                         Model S1                                                                              -164°                                                                      -50°                                                                      149°                                                                         3.17  3.55  2.72  1.39 1.35                              Model S2                                                                              -180°                                                                      -90°                                                                      144°                                                                         3.25  3.59  3.34  0.37 1.57                              Trypsin                                                                       Catalytic His57                                                                         71°                                                                       85°                                                                      170°                                                                         2.70  3.25  2.70  --   --                                (actual)                                                                      His P2 side                                                                   chain                                                                         Model T1                                                                              -155°                                                                      -79°                                                                      179°                                                                         2.78  4.78  3.28  0.98 2.10                              Model T2                                                                              -180°                                                                      -90°                                                                      158°                                                                         2.48  5.09  3.76  0.58 2.09                              __________________________________________________________________________

In general, modified substrates may be naturally occurring substratescontaining amino acid sequences which previously were not recognized bythe precursor enzyme or other enzymes or may be recombinant substrates.Thus, for example, in the former case the inventors have determined thatthe subtilisin mutant S24C:H64A is reactive with peptides correspondingto the naturally occurring substrates inhibin (between residues 61 and80) and ACTH (between residues 1 and 10). See Example 9 and Table VII.

In the latter case, the recombinant substrate is engineered to bereactive with a specific enzyme mutant. Such recombinant substratesinclude, for example, a recombinant polypeptide containing a prosequence (such as the Trp LE sequence from E. coli) and a desiredpolypeptide. Such recombinant polypeptides are typically generated tofacilitate the expression and/or secretion of the recombinantpolypeptide. However, in many instances, the sequence combined with thedesired polypeptide is not cleaved selectively from the recombinantpolypeptide upon secretion or by other known methods (e.g. by relativelynonspecific chemical reactions, such as treatment with CNBr,hydroxylamine, etc.).

This problem is overcome by the use of the enzyme mutants of the presentinvention with a "fusion polypeptide" which incorporates a targetcleavage sequence which is recognized by the mutant enzyme and whichwill assist in its own catalytic conversion to products. Thus, as usedherein, a fusion polypeptide is a recombinant polypeptide which containsan amino-terminal portion, a carboxyl-terminal portion and a targetcleavage sequence interposed between the amino and carboxyl terminalportions of the recombinant polypeptide. The target cleavage sequence isreactive with a particular mutant enzyme of the invention. Fusionpolypeptides may also be naturally occurring proteins that are mutatedto contain the target cleavage sequence. For example, protein A andprotein G bind to IgG. protein A, or its IgG binding domain, maytherefore comprise the amino terminal portion of a fusion polypeptidesuch that purification of the fusion polypeptide can be facilitated byaffinity chromatography. Thereafter, the amino terminal portion of thefusion polypeptide together with all or part of the target cleavagesequence is removed from the desired carboxy terminal portion bycleavage with a mutant enzyme specific for the target cleavage site.Other examples of amino and carboxyl terminal portions are listed inTable IV.

                  TABLE IV                                                        ______________________________________                                        Fusion Polypeptides                                                                          Target                                                         Amino-Terminal Cleavage    Carboxy-Terminal                                   Portion        Sequence    Portion                                            ______________________________________                                        Protein A      e.g, optimal                                                                              Surfactin(s)                                       Trp LE         P.sub.4 residues                                                                          Insulin A chain                                    β galactosidase                                                                         P.sub.3, P.sub.2, P.sub.1                                                                 Insulin B chain                                    Protein G      for H64A    Pro insulin                                        Ubiquitin      subtilisins Relaxin A chain                                    Maltose binding            Relaxin B chain                                    protein                    Pro relaxin                                                                   IGF-I                                                                         IGF-II                                                                        Brain IGF-I                                                                   DNase I                                                                       TGFα                                                                    TGFβ                                                                     Trigramin                                                                     tPA                                                                           γIfn, αIfn or βIfn                                           IL1                                                                           IL2                                                                           IL3                                                                           TNFα                                                                    EGF                                                                           NGF                                                                           Kistrin                                                                       CD4                                                                           Human growth                                                                  hormone                                            ______________________________________                                    

Fusion polypeptides preferably are constructed such that the targetcleavage sequence is accessible to the mutant enzyme without the need todenature the fusion polypeptide after it is expressed. Thus, the aminoterminal portion of the fusion polypeptide is chosen such that itsC-terminal amino acid is located on or near the surface. Similarly, thecarboxy terminal portion of the fusion polypeptide is preferably chosensuch that its amino terminal amino acid is on or near the surface of thepolypeptide forming the carboxy terminal portion. Thus, when the targetcleavage sequence is interposed between the amino terminal portion andcarboxy terminal portion of the fusion polypeptide, the target cleavagesequence is accessible to the mutant enzyme for catalysis.

In some instances, the particular construction of a fusion polypeptidemay not result in a fusion polypeptide containing an accessible targetcleavage sequence. In such cases, it is possible to denature the fusionpolypeptide, either partially or completely, to facilitate catalysis.Thereafter, the desired portion of the fusion polypeptide may berenatured. Such denaturation-renaturation methods are well known tothose skilled in the art.

In the case of enzyme mutants which do not act on polypeptidesubstrates, the modified substrate will consist of a substrate for aprecursor enzyme which has been appropriately modified to contain amodified moiety which is catalytic when in contact with the enzymemutant. Such modified substrates can be designed by substrate modelingas described above using the three-dimensional X-ray crystal structureof a precursor enzyme or enzyme-substrate complex. The construction ofsuch modified substrates, of course, will depend upon the chemicalnature of the modified substrate as determined by such modeling andcould involve biochemical and/or chemical modification or synthesis ofthe modified substrate.

In an alternate embodiment of the invention, the catalytic group removedfrom the precursor enzyme is not provided by a modified substrate.Rather, the catalytic group is provided by a catalytic cofactor whichcontains the catalytic group removed to form the enzyme mutant or anequivalent to that catalytic group (see Tables I and II). In such anembodiment, the enzyme mutant in conjunction with the catalytic cofactormaintains the same or similar specificity for substrate as the precursorenzyme.

In a specific embodiment, the subtilisin BPN' mutant S24C:H64A formed acatalytically active mutant enzyme-substrate-cofactor complex withspecific synthetic substrates and the catalytic cofactor imidazole. Inthe absence of imidazole, the S24C:H64A mutant had very low catalyticactivity with the synthetic substrates sAAPF-pna and sFAAF-pna. Cleavageof the substrates, however, was enhanced on the addition of imidazole.Thus, the catalytic function of the imidazoyl catalytic group ofhistidine which in this case has been replaced with alanine can beprovided by way of an appropriate catalytic group in a modifiedsubstrate or by way of an appropriate catalytic cofactor to render themutant enzyme catalytically active.

In the case of H64A subtilisin mutants used in conjunction with acatalytic cofactor such as imidazole, preferred substrates contain anamino acid sequence corresponding to amino acid residues P₄, P₃, P₂ andP₁ of substrates efficiently cleaved by subtilisin BPN'. Particularlypreferred P₁ residues include tyrosine, phenylalanine, methionine,leucine and lysine, most preferably phenylalanine and tyrosine. P₂residues may comprise any amino acid but are most preferably alanine,glycine, proline or serine. P₄ residues are preferably phenylalanine,isoleucine, methionine, alanine, leucine, lysine and valine, mostpreferably phenylalanine. Particular P₄ through P₁ amino acids sequencesuseful with H64A subtilisin mutants in conjunction with imidazoleinclude alanine-alanine-proline-phenylalanine andphenylalanine-alanine-alanine-phenylalaline.

The use of catalytic cofactors in conjunction with the mutant enzymes ofthe invention provides an exquisite means for controlling the catalyticactivity of the enzyme mutant with substrates. Thus, the enzyme mutantof the invention and a substrate (not comprising a modified substrate)can be combined without substantial catalysis occurring until thecatalytic cofactor is added.

In determining how a catalytic group should be replaced or modified in aprecursor enzyme, consideration must be given to the modified substrateor catalytic cofactor and substrate with which the enzyme is targeted tobe reacted with. In general, since the modified substrate will beproviding a catalytic group removed from the precursor enzyme, the aminoacid residue in the precursor enzyme should be replaced or modified insuch a way as to provide space for the modified moiety of the modifiedsubstrate. Typically, this requires that the side chain of the precursoramino acid be reduced in volume so that the enzyme mutant can receivethe moiety of the modified substrate.

The mean amino acid volume of amino acids when contained within aprotein and the mean side chain volume of such amino acids normalized toa zero side chain volume for glycine are shown in Table V. As shown inTables I and II, there are various preferred and alternate amino acidswhich may be substituted for specific catalytic residues within theactive site of a precursor enzyme. In each case, the amino acid beingsubstituted for a catalytic residue has a mean side chain volume whichis smaller than the side chain of the catalytic residue replace. Ingeneral, the catalytic amino acid residue should be replaced with anamino acid such that the mean side chain volume change upon making thesubstitution is sufficient to accommodate the catalytic group orequivalent catalytic group of the modified substrate as determinedempirically or by modeling studies. Thus, for example, the substitutionof His-64 for Ala increases the active site volume by approximately 75Å³ (101 Å-26 Å³). This increase in volume, however, is sufficient toaccommodate the histidine at residue P2 in a modified substrate whichhas a mean side chain volume of 101 Å³. Of course the detailed structureof the complex should be preferably checked by molecular modeling andideally by X-ray crystallography to ensure that the specific side chainscan interact favorably and that they are not sterically excluded evenwhen compatible by the simple volume considerations described above.

                  TABLE V                                                         ______________________________________                                                  Chothia.sup.(1) Mean                                                Amino     Amino Acid Volume                                                                            Mean Side Chain                                      Acid      in Protein (Å.sup.3)                                                                     Volume.sup.(2) (Å.sup.3)                         ______________________________________                                        Gly        66             0                                                   Ala        92            26                                                   Ser        99            33                                                   Cys       118            52                                                   Pro       129            63                                                   Thr       122            56                                                   Asp       125            59                                                   Val       142            76                                                   Asn       135            69                                                   Ile       169            103                                                  Glu       155            89                                                   Leu       168            102                                                  Gln       161            95                                                   His       167            101                                                  Met       171            105                                                  Phe       203            137                                                  Lys       171            105                                                  Tyr       207            141                                                  Arg       202            136                                                  Trp       238            172                                                  ______________________________________                                         .sup.(1) Chothia (1984) Ann. Rev. Biochem. 53, 537                            .sup.(2) Normalized to zero side chain volume for glycine.               

In addition to providing sufficient space for the catalytic group orequivalent catalytic group of the modified substrate, the side chainfunctionality of the catalytic residue replaced in the precursor enzymeshould be altered to facilitate the binding and catalytic activity ofthe modified substrate. For example, where the side chain of a catalyticamino acid residue in the precursor enzyme contains positively ornegatively charged polar groups these amino acids should be replaced ormodified to contain side chain which contain non-polar or unchargedpolar groups. Such substitutions are summarized in Tables I and II.

"Expression vector" refers to a DNA construct containing a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of said DNA in a suitable host. Such controlsequences include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites, and sequences which controltermination of transcription and translation. The vector may be aplasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, "plasmid" and"vector" are sometimes used interchangeably as the plasmid is the mostcommonly used form of vector at present. However, the invention isintended to include such other forms of expression vectors which serveequivalent functions and which are, or become, known in the art.

The "host cells" used in the present invention generally are procaryoticor eucaryotic hosts which preferably have been manipulated by themethods disclosed in EPO Publication No. 0130756 to render themincapable of secreting enzymatically active endoprotease. A preferredhost cell for expressing subtilisin is the Bacillus strain BG2036 whichis deficient in enzymatically active neutral protease and alkalineprotease (subtilisin). The construction of strain BG2036 is described indetail in EPO Publication No. 0130756 and further described by Yang, M.Y., et al. (1984) J. Bacteriol. 160, 15-21. Such host cells aredistinguishable from those disclosed in PCT Publication No. 03949wherein enzymatically inactive mutants of intracellular proteases in E.coli are disclosed. Other host cells for expressing subtilisin includeBacillus subtilis I168 (EPO Publication No. 0130756).

Host cells are transformed or transfected with vectors constructed usingrecombinant DNA techniques. Such transformed host cells are capable ofeither replicating vectors encoding the enzyme mutants or expressing thedesired enzyme mutant. In the case of vectors which encode a pre orprepro form of the enzyme mutant, such mutants, when expressed, aretypically secreted from the host cell into the host cell medium.

"Operably linked" when describing the relationship between two DNAregions simply means that they are functionally related to each other.For example, a presequence is operably linked to a peptide if itfunctions as a signal sequence, participating in the secretion of themature form of the protein most probably involving cleavage of thesignal sequence. A promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; a ribosome binding siteis operably linked to a coding sequence if it is positioned so as topermit translation.

The genes encoding the naturally-occurring precursor enzyme may beobtained in accord with the general methods described in EPO PublicationNo. 0130756 or by other method known to those skilled in the art. As canbe seen from the examples disclosed in EPO Publication No. 0130756, themethods generally comprise synthesizing labelled probes having putativesequences encoding regions of the enzyme of interest, preparing genomiclibraries from organisms expressing the enzyme, and screening thelibraries for the gene of interest by hybridization to the probes.Positively hybridizing clones are then mapped and sequenced.

The cloned enzyme is then used to transform a host cell in order toexpress the enzyme. The enzyme gene is then ligated into a high copynumber plasmid. This plasmid replicates in hosts in the sense that itcontains the well-known elements necessary for plasmid replication: apromoter operably linked to the gene in question (which may be suppliedas the gene's own homologous promotor if it is recognized, i.e.,transcribed, by the host), a transcription termination andpolyadenylation region (necessary for stability of the mRNA transcribedby the host from the hydrolase gene in certain eucaryotic host cells)which is exogenous or is supplied by the endogenous terminator region ofthe hydrolase gene and, desirably, a selection gene such as anantibiotic resistance gene that enables continuous cultural maintenanceof plasmid-infected host cells by growth in antibiotic-containing media.High copy number plasmids also contain an origin of replication for thehost, thereby enabling large numbers of plasmids to be generated in thecytoplasm without chromosomal limitations. However, it is within thescope herein to integrate multiple copies of the hydrolase gene intohost genome. This is facilitated by procaryotic and eucaryotic organismswhich are particularly susceptible to homologous recombination.

Once the precursor enzyme gene has been cloned, a number ofmodifications are undertaken to enhance the use of the gene beyondsynthesis of the naturally-occurring precursor enzyme. Suchmodifications include the production of recombinant precursor enzymes(as disclosed in EPO Publication No. 0130756) and the production ofenzyme mutants.

The following cassette mutagenesis method may be used to facilitate theconstruction and identification of the enzyme mutants of the presentinvention although other methods including site-directed mutagenesis maybe used. First, the gene encoding the enzyme is obtained and sequencedin whole or in part. Then the sequence is scanned for a point at whichit is desired to make a mutation of one or more amino acids in theexpressed enzyme. The sequences flanking this point are evaluated forthe presence of restriction sites for replacing a short segment of thegene with an oligonucleotide pool which when expressed will encodevarious mutants. Such restriction sites are preferably unique siteswithin the gene so as to facilitate the replacement of the gene segment.However, any convenient restriction site which is not overly redundantin the gene may be used, provided the gene fragments generated byrestriction digestion can be reassembled in proper sequence. Ifrestriction sites are not present at locations within a convenientdistance from the selected point (from 10 to 15 nucleotides), such sitesare generated by substituting nucleotides in the gene in such a fashionthat neither the reading frame nor the amino acids encoded are changedin the final construction. The task of locating suitable flankingregions and evaluating the needed changes to arrive at two convenientrestriction site sequences is made routine by the redundancy of thegenetic code, a restriction enzyme map of the gene and the large numberof different restriction enzymes. Note that if a convenient flankingrestriction site is available, the above method need be used only inconnection with the flanking region which does not contain a site.

Mutation of the gene in order to change its sequence to conform to thedesired sequence is accomplished by M13 primer extension in accord withgenerally known methods. Once the gene is cloned, the restriction sitesflanking the sequence to be mutated are digested with the cognaterestriction enzymes and a plurality of end termini-complementaryoligonucleotide cassettes are ligated into the gene. The mutagenesis isenormously simplified by this method because all of the oligonucleotidescan be synthesized so as to have the same restriction sites, and nosynthetic linkers are necessary to create the restriction sites.

In the disclosed embodiment, subtilisin was chosen as a model to testthe concept of substrate-assisted catalysis. In the hydrolysis ofpeptide bonds by subtilisin, His-64 acts as a catalytic base in theformation of an acyl-enzyme intermediate and as a catalytic acid in thesubsequent deacyclation step. Stroud, R. M., et al. (1975), Proteasesand Biological Control (Cold Spring Harbor Laboratory, New York), p. 13;Kraut, J. (1977) Ann. Rev. Biochem. 46, 331.

The catalytic triad of subtilisin is shown in FIG. 2. As can be seen,Ser-221, His-64 and Asp-32 are positioned to facilitate nucleophilicattack by the serine hydoxylate on the carbonyl of the scissile peptidebond. Several hydrogen bonds may also help to stabilize the transitionstate complex for the tetrahedral substrate intermediate. One hydrogenbond is between aspartate and the positively charged histidine, Nδ1.Kossiakoff, A. A., et al. (1981) Biochem. 20, 6462-6474. A secondhydrogen bond forms between the scissile amide nitrogen of the substrateand the (NεZ) proton on the histidine. A third set of hydrogen bondsforms between the enzyme and the oxyanion that is produced from thecarbonyl oxygen of the substrate. This latter set of hydrogen bonds isformed differently by the mammalian serine proteases and subtilisin. Afourth hydrogen bond appears to exist between the amide nitrogen of thepeptide bond between P-1 and P-2 and the carbonyl oxygen of Ser125.Specifically, X-ray crystallographic studies of chymotrypsin (Henderson,R. (1970) J. Mol. Biol. 54, 341 ) indicate that two hydrogen bonds formbetween the substrate oxyanion and two main-chain amide protons from theenzyme (Gly-193 and the catalytic Ser-195). Crystallographic studies ofsubtilisin (Robertus, et al. (1972) Biochem. 11, 4293-4303; Matthews, etal. (1975) J. Biol. Chem. 250, 7120-7126; Poulos, et al. (1976) J. Biol.Chem. 250, 1097-1103) show that two hydrogen bonds are also formed withthe oxyanion; one hydrogen bond donor is from the catalytic Ser-221main-chain amide while the other is from one of the NεZ protons of theAsn-155 side chain. See FIG. 2.

The model shown in FIG. 5 revealed that the delta and epsilon nitrogensof the histidine in position P2 in the modified substrate can besuperimposed within about an angstrom by the corresponding nitrogens ofthe catalytic His-64 (not shown in FIG. 4). This suggested that if thehistidine in the catalytic triad of subtilisin was replaced by analanine using site directed mutagenesis, a histidine from the substratemay substitute for the missing catalytic group in the mutant enzyme.

Maturation of the primary subtilisin gene product (preprosubtilisin) tosubtilisin in B. subtilis is believed to be mediated by autoproteolysisthat involves trace amounts of active subtilisin (Power, S. D., et al.(1986) Proc. Natl. Acad. Sci USA 83, 3096). The H64A mutation caused asevere reduction in secretion of mature subtilisin. However, it waspossible to process and subsequently purify the inactive H64A mutant byco-culturing B. subtilis cells harboring the H64A mutant gene with B.subtilis cells carrying an active subtilisin gene ("helper") or byadding wild-type subtilisin. A simpler strategy of culturing B. subtiliscells harboring the H64A mutant in the presence of purified wild-typesubtilisin BPN' (or an active mutant thereof) is also possible (Example4).

Stringent precautions were taken to ensure the purification of H64Asubtilisin away from "helper" subtilisin and any other contaminatingproteases. Firstly, the mutant subtilisin was expressed in the B.subtilis host BG2036 described in EPO Publication No. 0130756, that wasdeficient in chromosomal copies of the genes for alkaline protease(subtilisin) and neutral protease. Secondly, to minimize "helper"contamination the ratio of "helper" cells to H64A cells in thefermentation culture was adjusted to 1:1,000. Thirdly, a functionallysilent Ser-24→Cys mutation that is located on the surface of subtilisin(Wells, J. A., et al. (1986) J. Biol. Chem. 261, 6564) was introducedinto the H64A mutant. This accessible cysteine served as an affinityhandle for purification of the H64A mutant away from the non-cysteinecontaining "helper" on an activated thiol sepharose column. Finally, theactive "helper" subtilisin contained a functionally silent Ala-48→Glumutation that altered its electrophoretic mobility relative to S24C:H64Aon native and SDS polyacrylamide gels. After purification, the S24C:H64Amutant was judged to be greater than 99% pure by silver stained SDS(Morrissey, J. H. (1981) Anal. Biochem. 11, 307; Laemmli, U. K. (1970)Nature 227, 680) and native polyacrylamide gel electrophoresis (Example6). These purification procedures, including the use of a helpersubtilisin which is capable of electrophoretic separation from thesubtilisin mutant, are not necessarily required to practice the presentinvention.

As indicated, in addition to the S24C:H64A mutant subtilisin, othermutations were introduced into this subtilisin mutant to increasecatalytic activity. Mutations which increase the activity of wild-typesubtilisin BPN' were found to have an approximately additive effect whencombined (Wells, J. A., et al. (1987), Proc. Natl. Acad. Sci., 84,5167-5171). Transferring these mutations to the S24C:H64A enzyme gaveincremental improvements in activity, and so provided a useful strategyfor enhancing catalytic efficiency. Furthermore the preference for Tyrover Phe at the P1 position for wild-type subtilisin BPN' was also foundto hold for the S24C:H64A enzyme, suggesting that this is a usefulapproach for substrate optimization.

Although the results on wild-type subtilisin BPN' are qualitativelysimilar to those with the S24C:H64A enzyme, there are significantquantitative differences. For example, the G166A mutation gave a 4-foldimprovement toward the Phe substrate in the S24C:H64A enzyme yet onlyabout a 2-fold improvement in the wild-type enzyme. Conversely, the 3mutations (E156S:G169A:Y217L) had a larger effect upon wild-type thanupon the S24C:H64A enzyme. Furthermore, partitioning of the increase incatalytic efficiency into the k_(cat) and K_(m) terms were significantlydifferent (Table VI). These differences probably reflect subtle changesin substrate binding and/or catalytic mechanism between wild-type andthe H64A variant enzymes.

                                      TABLE VI                                    __________________________________________________________________________    Kinetic Analysis of Mutant Subtilisins* against N-succinyl-L-Phe-L-Ala-L-H    is-L-X-p-nitroanilide                                                         where X is Phe or Tyr, at pH 8.60 and (25 ± 0.2)° C.                                                                          P1                          Substrate                                         Pref-                       sFAHF-pna                sFAHY-pna                erence                      k.sub.cat K.sub.m                                                                             k.sub.cat /K.sub.m                                                                     k.sub.cat K.sub.m                                                                             k.sub.cat /K.sub.m                                                                     Tyr/                Enzyme  s.sup.-1  M     s.sup.-1 M.sup.-1                                                                      s.sup.-1  M     s.sup.-1 M.sup.-1                                                                      Phe**               __________________________________________________________________________    WT      .sup. 4.4 ± 0.5                                                                      17 ± 2                                                                           (2.6 ± 0.2) × 10.sup.5                                                        .sup. 5.1 ± 0.1                                                                       6.6 ± 0.6                                                                       (7.8 ± 0.5) ×                                                        10.sup.5 3.0                 G166A   .sup. 2.3 ± 0.1                                                                       4.6 ± 0.5                                                                       (5.1 ± 0.4) × 10.sup.5                                                        .sup. 4.1 ± 0.1                                                                      26 ± 2                                                                           (1.6 ± 0.1) ×                                                        10.sup.5 0.31                E156S:G169A:                                                                          (5.9 ± 0.3) × 10.sup.1                                                         20 ± 3                                                                           (3.0 ± 0.3) ×  10.sup.6                                                       (2.8 ± 0.1) × 10.sup.1                                                          6.3 ± 0.5                                                                       (4.4 ± 0.2) ×                                                        10.sup.6 1.5                 Y217L                                                                         E156S:G166A:                                                                          (3.8 ± 0.1) × 10.sup.1                                                          7.6 ± 0.8                                                                       (5.0 ± 0.4) × 10.sup.6                                                        (3.0 ± 0.1) × 10.sup.1                                                         41 ± 4                                                                           (7.5 ± 0.6) ×                                                        10.sup.5 0.15                G169A:Y217L                                                                   S24C:H64A                                                                             (2.1 ± 0.1) × 10.sup.-2                                                        340 ± 30                                                                         (6.2 ± 0.4) × 10.sup.1                                                        (9.9 ± 0.2) × 10.sup.-2                                                        210 ± 10                                                                         (4.8 ± 0.1) ×                                                        10.sup.2 7.7                 S24C:H64A:                                                                            (3.8 ± 0.1) × 10.sup.-2                                                        150 ± 10                                                                         (2.6 ± 0.1) × 10.sup.2                                                        (1.5 ± 0.1) × 10.sup.-2                                                        340 ± 50                                                                         (4.2 ± 0.5) ×                                                        10.sup.1 0.16                G166A                                                                         S24C:H64A:                                                                            (4.5 ± 0.1) × 10.sup.-2                                                        200 ± 20                                                                         (2.2 ± 0.2) × 10.sup.2                                                        (1.5 ± 0.1) × 10.sup.-1                                                        150 ± 10                                                                         (1.0 ± 0.1) ×                                                        10.sup.3 4.5                 E156S:G169A:                                                                  Y217L                                                                         S24C:H64A:                                                                            (8.3 ± 0.1) × 10.sup.-2                                                        120 ± 10                                                                         (6.7 ± 0.2) × 10.sup.2                                                        (7.9 ± 0.2) × 10.sup.-3                                                        270 ± 10                                                                         (2.9 ± 0.1) ×                                                        10.sup.1 0.043               E156S:G166A:                                                                  G169A:Y217L                                                                   __________________________________________________________________________     *Mutants are designated by the single letter code for the wildtype amino      acid followed by the residue number and then the amino acid replacement.      Multiple mutants are identified by listing the single mutant components       separated by colons (for example the double mutant Ser24→Cys,          His64→Ala is designated S24C:H64A).                                    **Calculated from the ratio of k.sub.cat /K.sub.m terms for the Tyr P1 an     Phe P1 substrate                                                         

The 4 mutations E156S:G166A:G169A:Y217L together enhance the activity ofthe wild-type subtilisin with sFAHF-pna by 19-fold. These same mutationsincrease the activity of the S24C:H64A enzyme with His P2 (sFAHF-pna;Table VI) by 11-fold.

Determination of the substrate specificity of H64A subtilisin BPN'variants is important in assessing their utility in site-specificproteolysis. Specificity determinants for subtilisin extend over atleast 6 residues (FIG. 4): 4 on the N-terminal side of the scissile bond(P4 through P1) and 2 on the C-terminal side (P1' and P2'). Substratespecificity data for the prototype S24C:H64A variant are summarized inTable VII. A histidine residue at P2 is apparently necessary but notsufficient for efficient polypeptide hydrolysis by S24C:H64A subtilisinBPN'. The P1 residue is also important in determining the efficiency of"substrate-assisted catalysis" by H64A variants, as shown by thequalitatively similar effects of mutants in the P1 pocket upon theactivity of wild-type and H64A subtilisin BPN' towards Phe P1 and Tyr P1substrates (Table VI). However, in addition to Phe P1 and Tyr P1,substrates containing P1 Met and P1 Leu are also expected to be reactivewith H64A mutants. Since Phe and Leu are favorable P1 residues forwild-type subtilisin BPN' (Estell, et al., Science 233 (1986) 659-663).In addition to the preference for histidine at P2 and a largehydrophobic residue at P1, all substrates identified to date that areefficiently cleaved by the S24C:H64A enzyme have Phe, Met or Ile at theP4 position. However, it is expected that substrates containing Ala,Leu, Val and Lys as P4 will also be reactive based on the reactivity ofwild-type subtilisin with substitutes containing these amino acids atP4. For wild-type subtilisin (and probably for H64A variants) the sidechain of the P3 residue is orientated away form the enzyme towardssolvent, and consequently there is limited specificity at this sub-site.

                  TABLE VII                                                       ______________________________________                                        Substrate Specificity of S24C:H64A                                            Variant of Subtilisin BPN'                                                    P4   P3    P2    P1  P1'   P2'  Substrate Reference                           ______________________________________                                        A. Efficiently Cleaved Substrates                                             F    A     H     Y   pna                  Table VI                            F    A     H     Y   [X]*  G              Table VIII                          F    A     H     Y   T     R    Z-AP fusion                                                                             FIG. 12                                                             protein   and 13                              I    N     H     Y   R     M    Inhibin   1                                                                   β-chain                                                                  (residues                                                                     61-80)                                        F    A     H     F   pna                  Table VI                            M    E     H     F   R     W    ACTH      1                                                                   (residues                                                                     1-10)                                         B. Substrates Not Detectably Cleaved**                                        T    L     H     L   V     L    Ubiquitin 1                                                                   (residues                                                                     62-76)                                        Y    E     H     F   E     N    BOP gene  1                                                                   product                                                                       (residues                                                                     68-86)                                        N    Q     H     L   C.sup.SO 3.sup.H                                                                    G    Bovine Insulin                                                                          1                                                                   B Chain                                                                       (Oxidized)                                    G    S     H     L   V     E    Bovine Insulin                                                                          1                                                                   B Chain                                                                       (Oxidized)                                    R    G     H     S   P     F    Inhibin β-chain                                                                    1                                                                   (residues                                                                     61-80)                                        ______________________________________                                         [X] all common Lamino acids except Pro and Ile                                (1) Carter, P. and Wells, J.A. , Science 237 (1987) 394-399                   **Only small peptide substrates where the potential cleavage site is          presumably accessible have been included.                                

                  TABLE VIII                                                      ______________________________________                                        Digestion of Succinyl-L-Phe-L-                                                Ala-L-His-L Tyr-L-[X]-L-Gly (.sup.˜ 0.6 μM)                          by S24C:H64A Subtilisin BPN' (3.6 μM)                                      at pH 8.0 and (37 ± 0.2)° C.                                                      Relative Cleaved Rates                                          P1' residue   No. KCl  +2 M KCl                                               ______________________________________                                        Pro           <0.1      ND*                                                   Ile           <0.1     <0.1                                                   Asp            1       14                                                     Glu            1       10                                                     Met            6       ND                                                     Phe            7       ND                                                     Leu            7       ND                                                     Gly            9       ND                                                     Ser           10       19                                                     Val           10       ND                                                     Ala           17       ND                                                     Tyr           21       27                                                     Gln           23       81                                                     Trp           24       ND                                                     His           26       ND                                                     Lys           30       45                                                     Thr           35       ND                                                     Asn           36       58                                                     Cys           40       ND                                                     Arg            43**    ND                                                     ______________________________________                                         *ND, not determined                                                           **This corresponds to an absolute rate of cleavage of 2 × 10.sup.-2     s.sup.-1.                                                                

The S24C:H64A enzyme has very broad substrate specificity on theC-terminal side of the cleavage site (P1' and P2'). This is desirablesince these residues represent the first 2 residues of the protein ofinterest in a fusion polypeptide. All residues at P1' (apart form Ileand Pro) allow efficient substrate hydrolysis (Table VIII). Fromproteolysis of protein and synthetic peptide substrates, sequencescontaining at least Trp, Arg, Met or Gly at P2' can be cleavedefficiently (Table VIIA). It is likely that the Pro P2' is unfavorablefor H64A subtilisin as it is for the wild-type enzyme. X-raycrystallography shows that the main-chain amide nitrogen and carbonyloxygen of the P2' residue make hydrogen bonds with the main-chaincarbonyl oxygen and amide nitrogen of Asn218, respectively (Robertus, J.D., et al. (1972), supra).

The major problem in achieving site-specific proteolysis is thatdigestion may not be limited to the designed target sequence. Evenfactor X_(a) which has a very narrow substrate specificity, occasionallycleaves at other sites besides the Ile-Glu-Gly-Arg target sequence(Nagai, K., et al. (1987), Methods Enzymol., 153, 461-481). Digestion ofthe Z-AP fusion protein (containing a synthetic IgG binding domain, atarget for a cleavage enzyme and E. coli alkaline phosphatase; seeExample 12,13) by S24C:H64A subtilisin and the multiple mutantS24C:H64A:E156S:G166A:G169A:Y217L is restricted entirely to the targetsequence, even though there are 12 other histidine residues present(Chang, C. N., et al. (1986), Gene, 44, 121-125; Nilsson, B., et al.(1987), Protein Engineering 1, 107-113). Seven of these histidines aresurrounded by unfavorable P4, P1 or P1' residues and 4 others areunavailable because the histidine is coordinated to Zn²⁺ in native AP(Sowadski, J. M., et al. (1985), J. Mol. Biol., 186, 417-433). Asidefrom the target sequence, only His87 in AP is in the context of afavorable amino acid sequence (Y T H⁸⁷ Y A L). However this site, andall of the other histidine residues present in AP, are at leastpartially buried in the 3-dimensional structure (Sowadski, J. M., et al.(1985), supra) making them unavailable for hydrolysis. In contrast toS24C:H64A and its variants, the wild-type enzyme rapidly cleaves theZ-AP fusion at 2 sites within 4 residues of the designed target followedby degradation of the AP product. This suggests that the regioncontaining the target is highly accessible.

Assessment of the frequency of naturally occurring sites for S24C:H64Asubtilisin rests upon attempting digestion of a large number of proteinsubstrates. Nine other globular proteins (hen egg white lysozyme, horsecytochrome c, horseradish peroxidase, bovine pancreatic ribonuclease,spinach ferredoxin, bovine catalase, bovine serum albumin, human serumalbumin and human tissue-type plasminogen activator) which collectivelycontain more than 80 histidine sites, were not cleaved by the S24C:H64Aenzyme under similar (native) conditions as described in Example 12,13for the digestion of Z-AP fusion protein. In contrast, all of theproteins tested were digested at many sites by wild-type subtilisin. Insome cases it may be necessary to denature the fusion protein in orderto make the target site accessible for cleavage. Digestion of humanserum albumin (contains 16 histidine residues) after reduction andcarboxymethylation, gave rise to limited proteolysis by the S24C:H64Aenzyme (not shown) at a rate <100-fold that for cleavage of the Z-APfusion protein.

Based upon the natural abundance of histidine in proteins (2%; Klapper,M. H. (1977) Biochem. Biophys. Res. Commun. 78 1018-1024) and good P1residues, Tyr, Phe, Leu, and Met (collectively about 22%), the frequencyof good cleavage sites that only satisfy the P2 and P1 dominant sequencerequirement is ˜0.5%. Thus, although other histidines may be present inthe product protein, very few are likely to contain satisfactorydeterminants at P4, P1 and P1' as well as being accessible forhydrolysis by H64A variant subtilisins.

Nevertheless if cleavage does occur at a significant rate at a siteadditional to the target site then it may be possible to overcome thisby judicious choice of enzyme from the H64A family of subtilisinvariants and of linker sequence. For example, if the offending site hasTyr P1, then one could use the S24C:H64A:E156S:G166A:G169A:Y217L variantin combination with a Phe P1 linker. This variant favors Phe over Tyr atP1 by 23-fold whilst retaining high catalytic efficiency for Phe P1(Table VI).

The S24C:H64A subtilisin BPN' variant satisfies most of the criteriaconsidered to be desirable for an ideal site-specific protease. It isexquisitely specific on the N-terminal side of the cleavage site and yetbroadly specific on the C-terminal side to allow specific cleavage of atarget linker. This enzyme can be recovered free of detectable proteasecontaminants in high yields (>30 mg/l in shake flasks). It resists avariety of protease inhibitors (including PMSF, EDTA, leupeptin andpepstatin) which permits their use during digestion to inactivateprotease contaminants that may be present in fusion proteinpreparations. This enzyme, like wild-type subtilisin (Wells, J. A., etal. (1986), J. Biol. Chem., 261, 6564-6570), is fully active inreductants or 0.1% (w/v) SDS or 0.1% (v/v) tween 20 and is moderatelyactive in denaturants (20% activity is retained in 2M urea and 10%activity in 2M guanidine hydrochloride against sFAHF-pna, FIG. 11) thatmay be required to solubilize the fusion protein or to make the targetsite accessible for cleavage. Activity of the penta-mutant(S24C:H64A:E156S:G169A:Y217L), is enhanced about 4-fold compared withthe prototype H64A enzyme with the Z-AP fusion protein, and for bothenzymes activity is enhanced ≈3-fold in the presence of 2M KCl and afurther 2-fold by performing the digests at 50° C. instead of at 37° C.The penta-mutant has been irreversibly immobilized on a solid supportvia the C 24 residue and retains the ability to cleave the Z-AP fusion,albeit at rates several fold slower than for the free enzyme in solution(see Example 12, 13, 15). This type of protease column eliminates theneed to purify the protease away from the cleaved products andfacilitates recycling of the protease. A final advantage to a proteasewhich is amenable to rational design such as subtilisin BPN' is thatthere is an extensive structural and functional data base (Wells, J. A.,et al. (1988), Trends in Biochemical Sciences, 13, 291-297) that can beutilized as need be for further modification of specificity determinantsat the P4, P1, P1' and P2' binding sites.

Example 1 Construction of helper subtilisin containing a functionallysilent A48E mutation.

The construction of pS4 is described in detail in EPO Publication No.0130756. This plasmid is depicted in FIG. 6. pS4 contains 4.5 kilobase(kb) of sequence derived from pBS42 (solid line) and 4.4 kb of sequencecontaining the B. amyloliquefaciens subtilisin gene and flankingsequences (dashed line). pBS42 was constructed as described in EPOPublication No. 0130756 and Band, L. and Henner, D. J. (1984) DNA 3,17-21. It was digested with BamHI and ligated with Sau3A partiallydigested chromosomal DNA from B. amyloliquefaciens (ATCC No. 23844) asdescribed in EPO Publication No. 0120756. pS4 was selected from thisgenomic library.

pS4-5, a derivative of pS4 made according to Wells, et al. (1983)Nucleic Acids Res. 11, 7911-7924, was digested with EcoRI and BamHI, andthe 1.5 kb EcoRI-BamHI fragment recovered. This fragment was ligatedinto replicative form M-13 mp9 which had been digested with EcoRI andBamHI (Sanger, et al., (1980) J. Mol. Biol. 143, 161-178; Messing, etal., (1981) Nucleic Acids Res. 9, 304-321; Messing, J. and Vieira, J.(1982) Gene 19, 269-276). The M-13 mp9 phage ligations, designated M-13mp9 SUBT, were used to transform E. coli strain JM101 (ATCC 33876) andsingle stranded phage DNA was prepared from a two mL overnight culture.An oligonucleotide primer was synthesized having the sequence

    5'-GTAGCAGGCGGAGAATCCATGGTTCC-3

The primer included the sequence of the subtilisin gene fragmentencoding amino acids 44 through 52 except that the codon 48 normallyencoding alanine was substituted with the codon GAA encoding glutamate;the serine codon at 49 (AGC) was also converted to TCC to introduce aconvenient NcoI site.

The primer (about 15 μM) was labelled with [³² P] by incubation with[γ³² p]-ATP (10 μL in 20 μL reaction) (Amersham 5000 Ci/mmol, 10218) andT₄ polynucleotide kinase (10 units) followed by non-radioactive ATP (100μM) to allow complete phosphorylation of the mutagenesis primer. Thekinase was inactivated by heating the phosphorylation mixture to 68° C.for 15 minutes.

The primer was hybridized to M-13 mp9 SUBT as modified from Norris, etal., (1983) Nucleic Acids Res. 11, 5103-5112 by combining 5 μL of thelabelled mutagenesis primer (.sup.˜ 3 μM), ¹⁸ 1 μg M-13 mp9 SUBTtemplate, 1 μL of 1 μM M-13 sequencing primer (17-mer), and 2.5 μL ofbuffer (0.3M Tris pH 8, 40 mM MgCl₂, 12 mM EDTA, 10 mM DTT, 0.5 mg/mlBSA). The mixture was heated to 68° C. for 10 minutes and cooled 10minutes at room temperature. To the annealing mixture was added 3.6 μLof 0.25 mM dGTP, dCTP, dATP, and dTTP, 1.25 μL of 10 mM ATP, 1 μL ligase(4 units) and 1 μL Klenow (5 units). The primer extension and ligationreaction (total volume 25 μL) proceeded 2 hours at 14° C. The Klenow andligase were inactivated by heating to 68° C. for 20 minutes. The heatedreaction mixture was digested with BamH1 and EcoRI and an aliquot of thedigest was applied to a 6 percent polyacrylamide gel and radioactivefragments were visualized by autoradiography. This showed the [³² p]mutagenesis primer had indeed been incorporated into the EcoRI-BamH1fragment containing the now mutated subtilisin gene.

The remainder of the digested reaction mixture was diluted to 200 μLwith 10 mM Tris, pH 8, containing 1 mM EDTA, extracted once with a 1:1(v:v) phenol/chloroform mixture, then once with chloroform, and theaqueous phase recovered. 15 μL of 5M ammonium acetate (pH 8) was addedalong with two volumes of ethanol to precipitate the DNA from theaqueous phase. The DNA was pelleted by centrifugation for five minutesin a microfuge and the supernatant was discarded. 300 μL of 70 percentethanol was added to wash the DNA pellet, the wash was discarded and thepellet lyophilized.

pBS42 was digested with BamH1 and EcoRI and purified on an acrylamidegel to recover the vector. 0.5 μg of the digested vector, 0.1 μg of theabove primer mutated EcoRI-BamHI digested subtilisin genomic fragment,50 μM ATP and 6 units ligase were dissolved in 20 μl of ligation buffer.The ligation went overnight at 14° C. The DNA was transformed into theB. subtilis host BG2036.

Example 2 Construction of H64A Mutant Subtilisin

The B. amyloliguifaciens subtilisin gene on a 1.5 kb EcoRI-BamHIfragment (Wells, J. A., et al., (1983) Nucleic Acids Res. 11, 7911-7925)was cloned into M13mp11 (Messing, J. and Vieira, J., (1982) Gene 19,269-276) to give M13mp11SUBT and single-stranded DNA was isolated(Carter, P., et al., (1985) in "Oligonucleotide site-directedmutagenesis in M13" Anglian Biotechnology Limited). The mutation H64Awas constructed using the synthetic oligonucleotide H64A (5' CAACAACTCGGAACTCAC 3') and the M13SUBT template using a previously describedmethod (Carter, P., et al., (1985) Nucleic Acids Res. 13, 4431-4443).The asterisks in HA64 denote mismatches to the wild-type sequence andthe underlined is a unique SacII restriction site.

The primer (H64A) was annealed to the single-stranded M13SUBT templateextended for 12 hrs. at 4° C. with DNA polymerase I (Klenow fragment) inthe presence of deoxynucleoside triphosphates and T4 DNA ligase (Carter,P., et al., (1985) Nucleic Acids Res. 13, 4431-4443 ). The M13heteroduplex DNA was then transfected directly into the E. coli host BMH71-18 mutL (Kramer, B., et al., (1984) Cell 38, 879-887) . Mutant phagewere identified by colony blot hybridization screening as previouslydescribed (Carter, P. J., et al., (1984) Cell 38, 835-840).

Putative H64A mutants were verified by dideoxy nucleotide sequencing(Sanger, F., et al., (1977) Proc. Natl. Acad. Sci. USA 77, 5463-5467) asmodified by Bankier, A. T. and Barrell, B. G., (1983) in "Techniques inthe life sciences" B5, Nucleic Acids Biochemistry, B508, 1, Elsevier,Ireland and designated M13mp11SUBT-Ala-64. The 1.5 kb EcoRI-BamHIfragment from M13mp11SUBT-Ala-64 was isolated and ligated with the 3.7kb EcoRI-BamHI fragment from the B. subtilis-E. coli shuttle vectorpBS42 (Band, L. and Henner, D. J., (1984) DNA 3, 17-21). E. coli MM294cells (Murray, N. E., et al., (1977) Mol. Gen. Genet. 150, 53) weretransformed with the ligation mixture using a CaCl₂ procedure (Cohen, S.N., Chang, A. C .Y., and Hsu, L., (1972) Proc. Natl. Acad. Sci. USA 69,2110-2114). Plasmid DNA was recovered from individual transformantsusing an alkaline-sodium dodecyl sulfate (SDS) procedure (Birboim, H. C.and Doly, J., (1979) Nucleic Acids Res. 7, 1513-1528 as modified byBurke, J. F. and Ish-Horowicz, D., (1982) Nucleic Acids Res. 10,3821-3830) to generate pBS42SUBT-Ala-64. The H64A mutation was verifiedby restriction endonuclease digests of the plasmid DNA using the enzymesSacII and BamHI which generate a 0.9 kb fragment.

Example 3 Construction of the double mutant S24C:H64A

The double mutant S24C:H64A was constructed from the single mutantspBS42SUBT-Cys-24 (Wells, J. A. and Powers, D. B., (1986) J. Biol. Chem.261, 6564-6570) and pBS42SUBT-Ala-64 (this document) by a 3-way ligationusing the following fragments: 3.7 kb EcoRI/BamHI from pBS42, 0.5 kbEcoRI/Clal from pBS42SUBT-Cys-24 and the 1.0 kb Clal/BamHI frompBS42SUBT-Ala-64. The double mutant Cys-24/Ala-64 was identified byrestriction endonuclease site markers introduced by the single mutations(His-64->Ala: new SacII site; Ser-24->Ala: Sau3A site removed) anddesignated pBS42SUBT-Cys-24/Ala-64. The pBS42SUBT-Cys-24/Ala-64 plasmidwas introduced into the B. subtilis host BG2036 (Anagostopoluos, C. andSpizizen, J., (1961) J. Bacteriol. 81, 741-746) deficient in alkalineand neutral proteases (Yang, M. Y., et al., (1984) J. Bacteriol. 160,15-21).

Example 4 Expression of S24C:H64A subtilisin BPN' by Co-culturing withA48E subtilisin BPN' or by culturing in the presence of purifiedwild-type subtilisin BPN'

Mutant subtilisin genes were expressed in BG2036 by fermentation inshake flasks using 2×TY media (Miller, J. H., (1972) in "Experiments inMolecular Genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) containing 12.5 μg/ml chloramphenicol at 37° C. for 18-20 hrs.Co-cultures were made by diluting S24C:H64A cultures 1:100 and A48Ecultures 1:100,000 in 2×TY containing 12.5 μg/ml chloramphenicol andgrown at 37° C. for 20-24 hrs with vigorous aeration.

An alternative procedure is to omit the co-culturing step above and toinclude purified wild-type subtilisin BPN' enzyme or the A48E enzyme, orsome other active subtilisin mutant enzyme in the culturing step. Asuitable amount of active subtilisin is 1 mg/l of the culture.

Example 5 Purification of S24C:H64A

Cultures were centrifuged (8,000 g, 15 min., 4° C.) and 3 volumes ofethanol (-20° C.) added to the supernatant. After centrifugation (8,000g, 15 min., 4° C.), the pellet was resuspended in 50 mM Tris.HCl (pH 8.0), 5 mM CaCl₂, 10 mM dithiothrietol (DTT), 0.1 mM phenylmethylsulfonylfluoride (PMSF) . After centrifugation (40,000 g, 30 min., 4° C.) thesupernatant was dialyzed against 21 10 mM 2-[N-morpholino]ethanesulfonicacid (MES) (pH 6.0), 5 mM CaCl₂, 10 mM DTT, 0.1 mM PMSF (S buffer)overnight at 4° C. The dialysate was passed over a 50 ml DE52 (Whatman)column and loaded on to 50 ml CM Trisacryl (LKB) column. Subtilisin waseluted with a 600 ml gradient of S buffer containing 0-100 mM NaCl at1.5 ml/min. Pooled subtilisin containing fractions were dialyzed against2 L deaerated 10 mM MES (pH 6.0), 5 mM CaCl₂, 100 mM NaCl, 0.1 mM PMSF(T buffer). Samples were loaded on to an activated thiol sepharosematrix (Pharmacia) washed extensively with T buffer and then eluted withT buffer containing 20 mM DTT. The eluate was concentrated usingCentricon 10 microconcentrators (Amicon) and then transferred to 10 mMMES (pH 6.0), 5 mM CaCl₂, 10 mM DTT, 0.1 mM PMSF (U buffer) by gelfiltration using PD10 G25 (Pharmacia) columns. The concentration ofsubtilisin was determined from the measured absorbance at 280 nm (E₂₈₀0.1%=1.17) (Matsubara, H., et al., (1965) J. Biol. Chem. 240,1125-1130). Aliquots of purified enzyme were flash frozen in liquidnitrogen and then stored at -70° C.

Example 6 Preparative native gel electrophoresis

1.5 mg S24C:H64A subtilisin in U buffer (defined in Example 5) wasadjusted to 10 mM phenyl boronate, 10% glycerol (v/v) and 0.1% (w/v)methylene blue. The sample was electrophoresed for 24 hrs. at 7 W(constant) on a 10% polyacrylamide gel (20 cm×20 cm×0.75 cm) withrecirculating buffer. The running buffer and gel contained 10 mM phenylboronate, 2 mM CaCl₂, 5 mM DTT 50 mM histidine and 60 mM3-[N-morpholino]propanesulfonic acid (MOPS). The protein wasdiffusion-blotted on to nitrocellulose (Hancock, K., and Tsang, V. C.W., (1983 Anal. Biochem. 133, 157-162 as modified by Carter, P., et al.,(1986) Proc. Natl. Acad. Sci. USA 83, 1189-1192). Subtilisin wasvisualized after binding rabbit anti-subtilisin antibody (Power, S.D.,et al., (1986) Proc. Natl. Acad. Sci. USA 83, 3096-3100) then horseradish peroxidase conjugated protein A by using the chromogenicsubstrate 3,3'-diaminobenzidine tetrahydrochloride. Subtilisincontaining gel slices were placed in dialysis bags with 6 ml runningbuffer (omitting phenyl boronate) and electroeluted at 10 mA (constant)for 20 hrs. at 4° C. Recovered material was concentrated and transferredto U buffer as for column purified enzyme (above).

Example 7 Kinetic analysis of S24C:H64A

The kinetic parameters for S24C and S24C:H64A were determined againstthe substrates N-succinyl-L-Phe-L-Ala-L-[X]-L-Phe-ρ-nitroanilide(abbreviated sFAXF-pna), where X (P2 position) was Ala, Gln, or His(Table IX). The kinetic parameters for the S24C enzyme are essentiallyidentical to wild-type subtilisin against these substrates indicatingthat the S24C mutation is kinetically silent. By comparison, the H64Amutation causes a drop of 10⁶ fold in k_(cat) /K_(m) against the Ala andGln P2 substrates. Almost all of the decrease in catalytic efficiency iscaused by a decreased k_(cat) term (up to 10⁶ times), although smallerbut significant increases appear in K_(m). Unlike wild-type or S24Csubtilisin, the S24C:H64A enzyme was completely resistant to inhibitionby the active site reagent, phenylmethylsulfonyl-fluoride (PMSF). Thissuggests the catalytic histidine is critical for stable sulfonylation byPMSF. Although the proportion of functional active sites in S24C:H64Aenzyme preparations could not be determined directly by such active sitelabeling, enzyme that was purified by additional native gelelectrophoresis (Example 6) had identical kinetic parameters toS24C:H64A described in Table IX.

The data are consistent with His-64 being extremely important incatalysis (presumably by proton transfer) and only marginally-importantin substrate binding. However, because we cannot be sure that acylationis rate limiting for the H64A mutant, as it is for the wild-type enzyme(Wells, J. A. (1986) Phil. Trans. R. Soc. Lond. A, 317, 415-423), therelatively small changes in K_(m) may not reflect changes in theenzyme-substrate dissociation constant (K_(s)) but rather a shift in therate determining step of the reaction (Guttreund, et al. (1956) BiochemJ., 63, 656) . In any case, removal of the catalytic imidazole causes areduction of about 10⁶ -fold in the total enzymatic rate enhancement(Table IX).

The catalytic efficiency (k_(cat) /K_(m)) of S24C toward the three P2substrates are all within a factor of five of each other. For theS24C:H64A mutant, k_(cat) /K_(m) for the Ala and Gln P2 substrates areessentially the same; however, hydrolysis of the HisP2 substrate is 170to 210 times more efficient, respectively. Essentially all of theincrease in k_(cat) /K_(m) for the His over the Ala and Gln P2substrates results from the k_(cat) term being larger by a factor of2,000 and 500, respectively. The larger K_(m) values for the His and GlnP2 substrates compared to Ala may reflect reduced binding affinityresulting from a bulky group at P2. Larger K_(m) values are alsoobserved for the Gln and His substrates for the S24C enzyme. Thus, thedrop in k_(cat) /K_(m) caused by the H64A mutation is partially restoredwhen cleaving a His P2 substrate. The net effect is a marked increase insubstrate preference for a His P2 side-chain brought about at the levelof catalysis rather than binding. The non-enzymatic hydrolysis rate ofthe HisP2 substrate is similar to the Ala and Gln P2 substrates (TableIX). Thus, the His P2 substrate only becomes functionally active in thecontext of the catalytic groups provided by the enzyme.

The fact that the catalytic efficiency of the S24C:H64A mutant againstthe His P2 substrate is 5,000 fold below wild-type suggests the His fromthe substrate P2 functions poorly in catalysis. This may result from theHis P2 making poor steric contacts and/or improper alignment of thecatalytic triad. Indeed, the model of the His P2 side-chain does notexactly match the catalytic His-64 in that the planes of the histidinesfrom the enzyme and substrate are almost perpendicular to each other(FIG. 5).

Example 8 pH Dependence of Amide Bond Hydrolysis by S24C:H64A Subtilisin

The pH dependence of k_(cat) /K_(m) for wild-type subtilisin shows asigmoidal increase from pH 6 to 8 (Glazer, A. N. (1967), J. Biochem.,242 433) that reflects the titration of the catalytic His64 (pK_(a)=7.1±0.1). The wild-type pH profile remains relatively flat over therange of 8-10 and declines thereafter (Ottesen, et al. (1970), InMethods of Enzymology (Ed. Perleman, Acad. Press, N.Y., Vol 19, p.199)).

FIG. 7 shows the pH dependence of hydrolysis of p-nitroanilide peptidesubstrates by S24C:H64A subtilisin. Analysis of S24C:H64A againstsFAAF-pna (FIG. 7A) was determined as in Table IX except using 100 mMTris.HCl or 100 mM 3-[cyclohexylamino]-1-propane sulfonic acid (CAPS)buffer. The data was fitted assuming a linear relationship withhydroxide ion concentration (solid lines in FIGS. 7A and 7b). Analysisof S24C:H64A with sFAHF-pna (FIG. 7C) was determined as in Table IXexcept using 100 mM 3-[N-morpholino] propanesulfonic acid (MOPS) buffer(filled circles) or 100 mM Tris.HCl (open circles) and then normalizingthe ionic strength using KCl. The data was fitted to a sigmoidrelationship (solid line) using a least-squares fit procedure.

The pH dependence of k_(cat) /K_(m) is markedly different for theS24C:H64A enzyme. For the sFAAF-pna substrate, there is an increase of15 fold in the k_(cat) /K_(m) between pH 8 to 10 (FIG. 7A). k_(cat)/K_(m) shows a linear dependence upon hydroxide ion concentration (FIG.7B) suggesting that a hydroxide ion can act as a catalytic base in theabsence of a catalytic histidine side chain. If one were to extrapolatefrom the increase in k_(cat) /K_(m) as a function of hydroxideconcentration (2×10⁴ s⁻¹ M⁻²), to the k_(cat) /K_(m) for S24C againstthis same Ala P2 substrate (8×10⁵ s⁻¹ M⁻¹), then the equivalentconcentration of the hydroxide ion would be about 40M.

In contrast, the k_(cat) /K_(m) for hydrolysis of the sFAHF-pna byS24C:H64A shows a sigmoidal pH dependence between pH 6 and 8 (FIG. 7C)that is similar to wild-type subtilisin. The pK_(a) of the activitydependent group is 6.8±0.1, and almost all of the pH dependent changesin k_(cat) /K_(m) result from changes in k_(cat) (data not shown). Forthe sFAHF-pna substrate, there is not a strong linear increase ink_(cat) /K_(m) with hydroxide above pH 8 as observed for hydrolysis ofsFAAF-pna. These data strongly suggest that the P2 histidine side-chainfrom the substrate can substitute functionally for the missing catalytichistidine 64.

The data presented in Table IX (measured at pH 8.6) underestimate thesubstrate preference for His over Ala (and Gln) because the k_(cat)/K_(m) for the sFAHF-pna is maximal at pH 8.0 (FIG. 7C), whereas for thesFAAF-pna substrate it is significantly lower at pH 8.0 (FIG. 7B). Thus,for S24C:H64A at pH 8.0, we estimate the substrate preference is up to400 times for the His P2 substrate over the corresponding Ala or Glnsubstrates.

TABLE IX

Kinetic analysis of mutant subtilisin against the substrates,N-succinyl-L-Phe-L-Ala-L-X-L-Phe-ρ-nitroanilide, where X is Ala, Gln, orHis. Six hydrolysis assays were performed simultaneously againstcorresponding substrate blanks in 0.10M Tris-HCl (pH 8.6), 10 mM DTT at25°±0.1° C. using a Kontron unvikon 860 spectrophotometer. Initialreaction rates were determined from the increase in absorbance caused bythe release of ρ-nitroaniline (ε_(M) ⁴¹⁰ =8,480 M⁻¹, cm⁻¹ (DelMar, E.G., et al. (1979) Anal. Biochem. 99 316)) and fitted by linearregression to an Eadie-Hofstee plot to calculate V_(max) and K_(m).k_(cat) was calculated from V_(max) /[enzyme], using thespectrophotometrically determined enzyme concentration (Matsubara, etal. (1965) J. Biol. Chem. 240, 1125). Enzyme concentrations in theassays were about 50 μg/mL for S24C:H64A and 1 μg/mL for S24C. Standarderrors in all determinations were below 20%. Slight variation in theabsolute kinetic values has been observed between batches of enzyme, butthe relative values among substrates has remained constant.

                                      TABLE IX                                    __________________________________________________________________________    Substrate                                                                     S24C             S24C:H64A     Non-enzymatic                                        k.sub.cat                                                                        K.sub.m                                                                          k.sub.cat /K.sub.m                                                                 k.sub.cat                                                                           K.sub.m                                                                          k.sub.cat /K.sub.m                                                                 hydrolysis rate                                P2 residue                                                                          s.sup.-1                                                                         μM                                                                            s.sup.-1 M.sup.-1                                                                  s.sup.-1                                                                            μM                                                                            s.sup.-1 M.sup.-1                                                                  (s.sup.-1)                                     __________________________________________________________________________    Ala   8.1                                                                              10 8.0 × 10.sup.5                                                               8.1 × 10.sup.-6                                                                32                                                                              0.25 1.7 × 10.sup.-7                          Gln   7.0                                                                              39 1.8 × 10.sup.5                                                               3.0 × 10.sup.-5                                                               150                                                                              0.20 7.1 × 10.sup.-8                          His   4.6                                                                              23 2.0 × 10.sup.5                                                               1.6 × 10.sup.-2                                                               380                                                                              42   7.9 × 10.sup.-8                          __________________________________________________________________________

Several lines of evidence indicate that the activity we attribute to theS24C:H64A enzyme is not the result of any other protease contamination.Firstly, the extreme substrate preference for His at the P2 position isunlike wild-type subtilisin or any known Bacillus protease. Secondly,the mutant has K_(m) values which are significantly different fromwild-type subtilisin suggesting differences in the energetics ofsubstrate binding and/or catalysis. Thirdly, the mutant is completelyresistant to inhibition by PMSF, unlike other serine proteases. In fact,the kinetic determinations for the S24C:H64A mutant are routinely madein the presence of PMSF to exclude any possibility of active "helper"subtilisin (Table IX, FIG. 7). Fourthly, the substrate dependent pHprofiles are unlike any protease we are aware of. Fifthly, preparationsof S24C:H64A are extremely pure from other contaminating proteins basedby analysis on SDS and native gels (>99%). Finally, the kinetic valuesdetermined for S24C:H64A that was additionally purified by native gelelectrophoresis (Example 6) are essentially the same as these reportedin Table IX.

Example 9 Hydrolysis of Polypeptide Substrates by S24C:H64A Subtilisin

To further evaluate the specificity of the S24C:H64A mutant incomparison with S24C, both enzymes were incubated with a 20 residuefragment of the inhibin β chain at pH 8.0 (Carter, P. and Wells, J. A.,Science (1987) 237 394-399). The choice of the peptide was based uponthe presence of two histidines (position 5 and 11) along with 16different amino acids, and a variety of large hydrophobic amino acidsthat are preferred amino acids at the P1 position of wild-typesubtilisin (Estell, D. A., .et al. (1986) Science 233, 659). FIG. 8shows the hydrolysis of the inhibin peptide substrateTVINHY↓RMRGHSPFANKLSC by S24C:H64A subtilisin. This substrate (100 μg)was digested with 10 μg S24C:H64A (FIG. 8A) or 0.13 μg S24C (FIG. 8B).Reaction mixtures were in a total volume of 250 μL containing 20 mMTris.HCl (pH 8.0), 10 mM dithiothreitol, 5% (v/v) dimethyl sulfoxide and1 mM PMSF (S24C:H64A only). After indicated times at 37° C., digestionproducts (monitored at 214 nm) were eluted from a reverse phase HPLCcolumn (Waters, C18) using a gradient (from left to right) of 0-50%(v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid .

After a 2-hour incubation with S24C:H64A (FIG. 8A), a ˜120 fold molarexcess of inhibin peptide (peak a) was cleaved to greater than 95%completion into two pieces (peaks b and c). Amino acid compositionanalysis of these two peptide fragments indicated cleavage had occurredbetween Tyr-6 and Arg-7, as expected for substrate assisted catalysis byHis-5 located at the P2 position from cleavage site. After 10-foldlonger digestion (20 hr.) a minor third peak appeared (labelled X inFIG. 8A). Analysis showed it to have the same composition as theundigested inhibin peptide. This minor product also appeared in anon-enzymatic blank incubation. No digestion was observed at the secondhistidine site.

In contrast to the two fragments produced by S24C:H64A the S24C enzymeproduced at least seven fragments (FIG. 8B) at a similar extent ofdigestion of starting material (compare 5 min. digestion with S24C to 2hr. digestion with S24C:H64A). Although none of these seven fragmentswere sequenced, the first two produced eluted from the HPLC profile atthe same positions as peaks b and c in FIG. 8A. Digestion to 95%completion of the starting peptide by S24C (30 min. incubation, FIG. 8C)produced more than ten different peptide fragments.

Example 10 Construction of Mutants of S24C:H64A Subtilisin

The mutations S24C:H64A:E156S:G169A:Y217L in the cloned Bacillusamyloliquefaciens gene (Wells, J. A., et al. (1983), Nucl. Acids Res.,11, 7911-7925) were constructed by ligating 3 fragments: 0.75 kbEcoRI/PvuI from pBS42SUBT-Cys-24/Ala-64 (Example 3) 0.75 kb PvuI/BamHIfrom the plasmid encoding E156S:G169A: Y217L (Wells, J. A., et al.(1987), Proc. Natl. Acd. Sci., 84, 5167-5171; EPO Publication No.035,652). The mutants S24C:H64A:G166A andS24C:H64A:E156S:G166A:G169A:Y217L were constructed by site-directedmutagenesis (Carter, P., et al. (1985), Nucleic Acids Res., 13,4431-4443) using a 36-mer oligonucleotide (5'GGTACCTCCGGG ACAGTGGCTACCCT 3' where * indicates mismatches and underline a new XhoI site)to install the G166A mutation. The templates used were M13mpllSUBT-Cys-24/Ala-64 and M13mpll SUBT-Cys-24/Ala-64/Ser-156/Ala-169/Leu-217 which are constructed from correspondingmutant subtilisin genes in the vector pBS42 (described above) in amanner analogous to Example 2. (The G166A mutation is introduced aspreviously described in Wells, et al. (1987), supra and EPO PublicationNo. 035,652.) Putative S24C:H64A:G166A and S24C:H64A:E156S:G166A:G169A:Y217L mutants in M13 were verified and then recloned into thevector pBS42 in a manner analogous to Example 2 and designatedpBS42SUBT-Cys-24/Ala-64/Ala-166 andpBS42SUBT-Cys-24/Ala-64/Ser-156/Ala-166/Ala-169/Leu-217, respectively.These mutants were transformed into the B. Subtilis host BG2036 asdescribed in Example 3 and then expressed and purified as described inExamples 4 and 5.

Example 11 Analysis of S24C:H64A Subtilisin and Mutants Thereof

Mutant enzymes were assayed with substrates,N-succinyl-L-Phe-L-Ala-L-His-L-X-p-nitroanilide, where X is either Pheor Tyr (sFAHF-pna and sFAHY-pna, respectively) , against correspondingblanks in 1 ml 100 mM Tris.HCl at pH 8.60, 4% (v/v) dimethyl sulfoxide(Me₂ SO) at (25±0.2)°C. with a Kontron Uvikon 8 60 spectrophotometer.Initial reaction rates were determined from the increase in absorbanceat 410 nm on release of ρ-nitroaniline (ε₄₁₀ =8,480 M⁻¹ cm⁻¹ ; Del Mar,E. G., et al (1979) Anal. Biochem. 99, 316-320) and fitted to theMichaelis-Menten equation using a least-squares fit procedure (Carter,P., et al. (1988) Nature 332, 564-568 ). Enzyme concentrations(determined spectrophotometrically; ε_(Z80) ⁰.1 %=1.17, Matsubara, H.,et al. (1965) J. Biol. Chem. 240, 1125-1130) in the assays were 1-20 nMfor H64 containing enzymes and 0.3-2 μM for H64A containing enzymes. Thesubstrate concentrations were determined after total hydrolysis andcorrected for background hydrolysis and were in the range of 0.1 K_(m)to 10 times K_(m).

The S24C:H64A enzyme (1.9 μM) was assayed with the substrate sFAHF-pna(200 μM) as described above (except that the concentration of Me₂ SO was1% (v/v)) in the presence of varying amounts of KCl, NaCl, guanidinehydrochloride, urea, SDS, sodium deoxycholate, nonidet P-40 or tween 20.

Peptide substrates (.sup.˜ 0.6 mM) having the formN-succinyl-L-Phe-L-Ala-L-His-L-Tyr-L-[X]-L-Gly (where [X] represents the20 common amino acids) were digested by S24C:H64A subtilisin (3.6 mM) in1 ml 20 mM Tris.HCl at pH 8.0, 1 mM dithiothreitol (DTT), 0.1 mMphenylmethylsulfonyl fluoride (PMSF), 3.5% (v/v) Me₂ SO, in the presenceor absence of 2M KCl at 37° C. At various times digests were applied toa C18 reverse phase HPLC column (Waters) and eluted with a gradient of 0to 40% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid. Elution ofthe substrate and N-succinyl-L-Phe-L-Ala-L-His-L-Tyr product fragmentwere monitored at 280 nm and quantified by peak integration and aminoacid composition analysis. The relative (and absolute) cleavage rate foreach peptide substrate was estimated from the initial rate of productformation in 4-6 successive time points.

Example 12 Construction, Expression, Purification and Digestion of Z-APFusion Protein

A phagemid pZAP encoding the Z-AP fusion protein was constructed byligating 3 fragments: 4.4-kilobase HindIII (filled-in)-NarI from proteinA phagemid vector pEZ (Nilsson, B., et al. (1987), Protein Engineering,1, 107-113), 1.4-kilobase NotI (filled-in)-MluI from the E. colialkaline phosphatase (AP) gene (Chang, C. N., et al. (1986), Gene, 44,121-125) engineered with sites for MluI and NotI (3' to coding sequence)and a synthetic cassette coding for a histidine-containing linker withMluI and NarI compatible ends. See FIG. 12. The ligation mixture wastransformed into E. coli JM101 and plated on to LB plates containing thechromogenic substrate for AP (5-bromo-4-chloro-3-indolyl phosphate; 2mg/ml). Several AP expressing clones (blue colonies) were verified bydideoxy sequence analysis (Sanger, F., et al. (1977), Proc. Natl. Acad.Sci. USA, 74, 5463-5467).

The Z-AP fusion was expressed in E. coli JM101 containing pZAP, purifiedfrom osmotically shocked cells by binding to IgG sepharose, and elutedwith lithium diiodosalicylate Nilsson, B., et al. (1985), EMBO J., 4,1075-1080). The purified Z-AP fusion protein was desalted by gelfiltration (PD10 disposable columns, Pharmacia) and then dialyzed at 4°C. overnight against 2 liters 50 mM Tris.HCl at pH 8.0. Aliquots wereflash frozen and stored at -70° C. Samples of the Z-AP fusion proteinthat were digested by mutant subtilisins (as described for FIG. 13),were precipitated with 10% (w/v) trichloroacetic acid and analyzed bySDS-PAGE (Laemmli, U.K. (1970), Nature, 227, 680-685). The AP digestionproduct (M_(r) =47,000) was electroblotted on to polyvinylidenedifluoride membrane (Matsudaira, P. (1987), J. Biol. Chem., 262,10035-10038) and the N-terminus was sequenced directly.

Example 13 Effect of Subtilisin Mutants on Catalytic Efficiency

A. Enhancing the Catalytic Efficiency of Subtilisin

The mutations G166A (Estell, D. A., et al. (1986), Science., 233,659-663) and E156S:G169A:Y217L (Wells, J. A., et al. (1987), Proc. Natl.Acad. Sci. USA, 84, 5167-5171) which enhance the activity of wild-typesubtilisin BPN' towardsN-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-ρ-nitroanilide (sAAPF-pna), wereexamined first singly and then in combination using the substratesFAHF-pna (Table VI, supra). The catalytic efficiency, k_(cat) /K_(m),of wild-type subtilisin with sFAHF-pna was increased 2 and 12-fold bythe mutations G166A and E156S:G169A:Y217L, respectively, and by 19-foldby combining these enhanced activity mutants.

Wild-type subtilisin hydrolyses the Tyr P1 substrate sAAPY-pna moreefficiently than the homologous Phe P1 substrate (sAAPF-pna) (Estell, D.A., et al. (1986), Science, 233, 659-663). Accordingly, both thewild-type and the E156:G169A:Y217L enzymes showed increases in k_(cat)/K_(m) towards sFAHY-pna compared to sFAHF-pna. As expected, the G166Aenzyme, which has been previously shown to sterically exclude asAAPY-pna substrate (Estell, D. A., et al. (1986), supra), is similarlyreduced on the sFAHY-pna substrate. This was also found for the mutantE156S:G166A:G169A:Y217L.

The combined effects of these binding site mutants were then evaluatedin the context of the H64A mutation with Phe P1 and Tyr P1 substrates(Table VI). A surface accessible thiol (S24C) that has no effect uponenzyme activity (Carter, P., et al. (1987), Science, 237, 394-399), wasintroduced into all H64A variant subtilisins to facilitate theirpurification. Numerous control studies (Carter, P., et al. (1987),supra; Carter, P., et al. (1988), Nature, 332, 564-568) havedemonstrated that the purified active site mutants are free ofdetectable contaminating protease activity. The G166A variant or thecombination of the 3 mutations (E156S:G169A:Y217L) improved k_(cat)/K_(m) by about 4-fold each. When all 4 mutations were introduced intothe S24C:H64A enzyme, k_(cat) /K_(m) was increased by more than 11-fold.The results with the Tyr P1 substrate toward the S24C:H64A family ofenzymes parallel also those with the wild-type family; theS24C:H64A:E156S:G169A:Y217L variant was the most efficient catalystfollowed by the S24C:H64A enzyme. Variants containing the G166A mutationwere actually much worse due to steric hindrance for the Tyr P1substrate as expected. Thus, the best enzyme-substrate pair within thisfamily is the penta-mutant (S24C:H64A:E156S:G169A:Y217L) subtilisin withthe sFAHY-pna substrate; this enzyme is nearly 20-fold improved over theprototype pair (S24C:H64A hydrolyzing the sFAHF-pna substrate).

B. Survey of Conditions for Hydrolysis Assay for S24C:H64A Subtilisin

The prototype S24C:H64A enzyme was assayed under a variety of conditionsto assess their use in digestion of protein substrates (FIG. 11). Highconcentrations of salt enhance the activity of the S24C:H64A enzyme(FIG. 11A), as has been shown for the wild-type enzyme (Otteson, M., etal. (1970), Compt. Rend. Tray. Lab. Carlsberg, 38, 369-383). TheS24C:H64A variant retains 50% of its activity in the presence of 1M ureaor 0.5M guanidine hydrochloride (FIG. 11B). The S24C:H64A variant isactive in non-ionic (tween 20 and nonidet P-40) and ionic (SDS andsodium deoxycholate) detergents and at concentrations that arefrequently used to solubilize and denature most proteins (eg, 0.1% (w/v)SDS). There was no detectable loss of activity during the kinetic runs(up to 30 min) under any of these conditions.

C. P1' Specificity of S24C:H64A Subtilisin

Specificity determinants for subtilisin BPN' extend for 2 residues onthe C-terminal side of the scissile bond (P1' and P2', FIG. 10), whichrepresent the first 2 residues of the protein of interest in a fusionprotein. The P1' specificity of the S24C:H64A enzyme was studied usingthe family of peptide substrates:N-succinyl-L-Phe-L-Ala-L-His-L-Tyr-L[X]-L-Gly, where X represents the 20common amino acids (Table VI, supra). The Phe-Ala-His-Tyr sequence waschosen as the most favorable p-nitroanilide substrate that we haveidentified for S24C:H64A subtilisin; glycine at P2' was chosen tominimally satisfy the P2'-enzyme main chain interactions. The relativerates of peptide cleavage were measured by the rate of formation of theproduct. N-succinyl-L-Phe-L-Ala-L-His-L-Tyr (Table VIII). In every casehydrolysis occurred exclusively after the Tyr residue as expected. Allof the P1' substrates were hydrolyzed at rates within 7-fold of eachother except for those containing Asp or Glu, which were cleaved slowly,or Pro or Ile which were not cleaved at detectable rates. Hydrolysis ofAsp or Glu P1' substrates was stimulated 10-fold by addition of 2M KCland cleavage of other substrates tested was increased 1.5 to 3.5-fold.

D. Specific Cleavage of a Model Fusion Protein

A model fusion protein was constructed (FIG. 12) that contains onesynthetic (Z) domain of Staphylococcus aureus protein A (Nilsson, B., etal. (1987), Protein Engineering, 1, 107-113), followed by the optimizedhistidine-containing linker (Phe-Ala-His-Tyr) and E. coli alkalinephosphatase (AP). See Example 12. In an attempt to improve theaccessibility of the site for cleavage, the target linker was precededby the sequence Pro-Gly, where the glycine replaces a trypsin-sensitivelysine in protein A (Sjodahl, J. (1977), Eur. J. Biochem., 78, 471-490).Furthermore, the N-terminus of AP is susceptible to proteolysis by bothtrypsin (Roberts, C. H., et al. (1984), J. Biol. Chem., 259, 729-733)(between Arg11 and Ala12) and by V8 protease (Tyler-Cross, R., et al.(1989), J. Biol. Chem., 264, 4523-4528) (between Glu9 and Asn10). AP wasalso an attractive marker protein because a similar fusion protein wasexpressed in high yield in the periplasmic space of E. coli with verylittle proteolytic degradation (Nilsson, B., et al. (1985), EMBO J., 4,1075-1080) and AP can be readily assayed using chromogenic substrates.The fusion protein was designed so that cleavage at the target sitegenerates AP with an additional N-terminal Thr residue. This sequence(containing a Mlu I site) was particularly convenient to construct, andsimplifies any subsequent manipulations to substitute the P1' residue.The protein A derived fusion protein was efficiently purified by IgGaffinity chromatography as previously described (Nilsson, B., et al.(1985), supra).

Protease digestion experiments (FIG. 13) show that the prototype enzyme(S24C:H64A) and the most active penta-mutant variant(S24C:H64A:E156S:G169A:Y217L) cleave the fusion protein (M_(r) =54,000)efficiently and specifically to generate a protein with the expectedelectrophoretic mobility of AP (M_(r) =47,000). The protein A fragment(M_(r) ≈7,000) is too small to be resolved from the dye-front on thisgel. N-terminal sequence analysis of the purified AP product in eachcase gave the sequence expected for cleavage at the designed target site(Thr-Arg-Thr-Pro-Glu-Met-Pro). Digestion by the penta-mutant was about4-fold faster than by the prototype H64A variant, and in each case thecleavage rate was enhanced about 3-fold by 2M KCl as was observed formany of the peptide substrates (Table VIII).

Example 14 Cleavage of Z-bIGF-I Fusion Protein byS24C:H64A:E156S:G169A:Y217L Subtilisin (bIGF-I=brain insulin-like growthfactor one)

A phagemid pZbIFG-I encoding the Z-bIGF-I fusion protein was constructedin a manner analogous to that for pZAP (Example 12) by ligating 3fragments: 4.4-kilobase HindIII (filled-in)-NarI from protein A phagemidvector pEZ (Nilsson, B., et al. (1987), supra, 0.25-kilobase NotI(filled-in)-MluI from bIGF-I engineered with sites for MluI and NotI (3'to coding sequence) and a synthetic cassette (exactly as in FIG. 12)coding for a histidine-containing linker with MluI and NarI compatibleends. See FIG. 14. The ligation mixture is transformed into E. coliJM101 and several clones are verified by dideoxy sequence analysis(Sanger, F., et al. (1977), Proc. Natl. Acad. Sci. USA, 74, 5463-5467).The Arg residue at the 2nd position of bIGF-I (introduced when the MluIsite was installed) is converted back to the natural residue (leucine)by site-directed mutagenesis (Carter, P. 1985 supra).

The Z-bIGF-I fusion protein was expressed in E. coli JM101 containingpZbIGF-I, purified from osmotically shocked cells by binding to IgGsepharose, and eluted with 1M acetic acid.

Aliquots of Z-bIGF-I were lyophilized, resuspended in distilled waterand re-lyophilized. After resuspending in distilled water, aliquots wereflash frozen and stored at -70° C. The Z-bIGF-I fusion protein wasdigested with S24C:H64A:E156S:G169A:Y217L subtilisin BPN' under the sameconditions as used for the Z-AP fusion protein (Examples 12, 13 and asdescribed for FIG. 13). The digestion products were analyzed by reversephase HPLC as described in Example 9. Collected peaks were lyophilizedand analyzed by mass spectroscopy (FIG. 15). The observed mass/charge(m/z) ratio (7366.4) for bIGF-I isolated after cleavage of Z-bIGF-I wasin excellent agreement with the theoretical value (7366.41). FurthermoreZ-bIGF-I was very efficiently cleaved by the S24C:H64A:E156S:G169A:Y217Lsubtilisin BPN' variant without having to reduce the 3 disulfide bridgespresent in the b-IGF-I molecule.

Example 15 Activity of S24C:H64A:E156S:G169A:Y217L Subtilisin BPN' AfterImmobilization on a Solid Support

The penta-mutant subtilisin variant S24C:H64A:E156S:G169A:Y217L wasimmobilized on to thiopropyl-sepharose 6B (Pharmacia) (the same solidsupport as used in Example 5) as follows:

1) The S24C:H64A:E156S:G169A:Y217L enzyme (60 nmol) was activated at theCys24 thiol with 1.5 μmol N,N'-1,4-phenylenedimaleimide in the presenceof 100 mM Tris-HCl (pH 7.5), 5 mM CaCl₂, for 1 hr at 4° C. Excesscross-linking reagent was then removed by gel filtration.

2) The thiopropyl-sepharose 6 B resin was deprotected by washing with 20mM DTT in 100 mM Tris HCl (pH 8.0), and then equilibrated with 100 mMTris-HCl (pH 7.5), 5 mM CaCl₂.

3) The deprotected thiopropyl-sepharose 6B resin was then mixed gentlywith the activated enzyme from (1) above (overnight, 4° C.). The resinwas equilibrated with 100 mM Tris-HCl (pH 8.0), and then remaining freethiol groups on the support blocked with 100 mM iodoacetamide in 100 mMTris HCl (pH 8.0) (2 hr, 4° C.). Finally, the resin was washedextensively with 100 mM Tris-HCl (pH 7.5), 5 mM CaCl₂.

4) The loading of enzyme on the resin was estimated from the knownamount of S24C:H64A:E156S:G169A:Y217L enzyme (60 nmol) used for couplingand that which remained in solution after the coupling step (estimatedfrom the enzyme activity).

Immobilized S24C:H64A:E156S:G169A:Y217L subtilisin BPN' was found tocleave the Z-AP fusion protein, albeit a few-fold slower than insolution.

Immobilization of subtilisin BPN' via Cys-24 provides a unique anddefined attachment point to a solid support. Furthermore, Cys-24 islocated on the surface of the enzyme distant from the active site (FIG.16). Thus immobilization of subtilisin BPN' via Cys-24 should notcompromise the accessibility of the active site of the enzyme even tomacromolecular substrates such as proteins.

The immobilization of enzymes has been extensively described in theliterature (see Methods in Enzymology, 135, 136, 137).

Furthermore, numerous immobilized proteases are commercially available,including the following taken from the 1989 Catalogue of the SigmaChemical Company: subtilisin BPN', bovine trypsin and α-chymotrypsin,carboxypeptidase A, papain, pepsin, proteinase K, thermolysin,Streptomyces griseus protease, and Staphylococcus aureus V8 protease.

These immobilized proteases are highly active even againstmacromolecular substrates. Indeed the activity units used by Sigma areusually based upon hydrolysis of the milk protein, casein.

Example 16 Stimulation of the Activity of S24C:H64A Subtilisin BPN' byImidazole

The S24C:H64A variant of subtilisin BPN', and also the wild-type enzyme,were assayed with the substrateN-succingl-L-Ala-L-Ala-L-Pro-L-Phe-P-nitroanilide (sAAPFpna) in 1 ml 100mM Tris-HCl (pH 8.60) 4% (v/v) dimethyl sulfoxide at 25°±0.2° C. with aKontron Uvikon spectrophotometer, by the method of initial rates asdescribed by Carter, P., et al. (1988), Nature, 332, 564-568, exceptthat the assays included varing concentrations of imidazole and theionic strength was kept constant by adjusting with NaCl. For theS24C:H64A enzyme k_(cat) with sAAPFpna was increased in the presence ofimidazole (Table XI) reaching a maximal 4-fold enhancement in thepresence of 300 mM imidazole.

K_(m) also increased with increasing imidazole so that at high imidazoleconcentrations k_(cat) /K_(m) actually decreased slightly. In contrastto the S24C:H64A variant, k_(cat) for the wild-type enzyme withsAAPF-pna was relatively unaffected by imidazole concentrations up to900 mM (Table X). K_(m) for the wild-type enzyme increased almost10-fold in the presence of 900 mM imidazole (Table X).

Thus, hydrolysis of the non-histidine substrate (sAAPF-pna) by S24C:H64Asubtilisin can be stiumlated by the addition of an exogenous generalbase, in this case imidazole.

                  TABLE X                                                         ______________________________________                                        Kinetic Analysis of wild-type subtilisin BPN' against                         sAAPF-pna at pH 8.60 and (25 ± 0.2)° C. in the presence             of varying concentrations of imidazole.                                       [Imidazole]                                                                             k.sub.cat   K.sub.m   k.sub.cat /K.sub.m ×                    mM        S.sup.-1    mM        10.sup.5 s.sup.-1 M.sup.-1                    ______________________________________                                        909       66.1 ± 1.4                                                                             2.0 ± 0.1                                                                            0.33 ± 0.01                                795       65.4 ± 1.0                                                                             1.5 ± 0.1                                                                            0.44 ± 0.01                                682       71.4 ± 1.2                                                                             1.3 ± 0.1                                                                            0.56 ± 0.01                                568       65.2 ± .19                                                                             0.95 ± 0.08                                                                          0.69 ± 0.01                                455       72.4 ± 2.3                                                                             0.74 ± 0.06                                                                          0.98 ± 0.1                                 318       64.3 ± 1.2                                                                             0.54 ± 0.03                                                                          1.2 ± 0.1                                  182       65.1 ± 1.0                                                                             0.39 ± 0.02                                                                          1.7 ± 0.1                                   0        57.6 ± 0.7                                                                             0.21 ± 0.01                                                                          2.8 ± 0.1                                  ______________________________________                                    

                  TABLE XI                                                        ______________________________________                                        Kinetic Analysis of S24C:H64A variant of subtilisin                           BPN' against sAAPF-pna at pH 8.60 and (25 ± 0.2)° C. in             the presence of varying concentrations of imidazole.                          [Imidazole]                                                                             k.sub.cat ×                                                                           K.sub.m  k.sub.cat /K.sub.m                           mM        10.sup.-4 s.sup.-1                                                                          mM       s.sup.-1 M.sup.-1                            ______________________________________                                        909       2.8 ± 0.1  8.9 ± 0.7                                                                           3.1 ± 0.2                                 454       2.8 ± 0.1  4.2 ± 0.3                                                                           6.6 ± 0.4                                 318       2.8 ± 0.1  3.3 ± 0.3                                                                           8.9 ± 0.6                                 200       2.0 ± 0.1  3.1 ± 0.3                                                                           6.6 ± 0.5                                 100       1.5 ± 0.1  1.9 ± 0.2                                                                           7.8 ± 0.8                                  50       1.2 ± 0.2  2.5 ± 0.3                                                                           4.5 ± 0.2                                  0        0.65 ± 0.03                                                                              1.8 ± 0.2                                                                           3.6 ± 0.3                                 ______________________________________                                    

The effect of imidazole on hydrolysis of the non-histidine containingsubstrate sFAAF-pna by S24C:H64A subtilisin BPN' was investigatedexactly as described for the sAAPF-pna substrate above.

FIG. 17 depicts the imidazole dependence of hydrolysis of sFAAF-pna atpH 8.60 and (25±0.2)°C. by S24C:H64A subtilisin BPN'.

k_(cat) for sFAAF-pna with the S24C:H64A enzyme was increased ˜20-foldin the presence of 750 mM imidazole (FIG. 17) and K_(m) was increased˜10-fold.

Thus the function of a missing catalytic group (in this case, histidine)can be restored at least partially by supplying an equivalent functionalmoiety (in this case, the imidazole moiety) exogenously.

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed .embodiments and that suchmodifications are intended to be within the scope of the presentinvention.

All literature references are expressly incorporated herein byreference.

What is claimed is:
 1. A process comprising contacting a subtilisin-related protease variant and a modified substrate wherein said variant is not found in nature and is formed by the replacement in a precursor subtilisin-related protease of a catalytic histidine with an amino acid having a side-chain volume less than the side chain volume of histidine, said histidine being catalytically functional when in contact with a selected region of a substrate, wherein said variant is substantially less active catalytically with said substrate as compared to said catalytic activity of said variant with at least one modified substrate, said modified substrate being formed by replacing an amino acid residue in said selected region with histidine.
 2. The process of claim 1 wherein said subtilisin-related protease is subtilisin.
 3. The process of claim 2 wherein said replaced or modified amino acid residue in said subtilisin is His-64 in B. amyloliquefaciens subtilisin.
 4. The process of claim 3 wherein said His-64 is replaced by Ala.
 5. The process of claim 3 wherein said modified substrate contains histidine at residue P2 of said modified substrate.
 6. The process of claim 2 wherein said modified substrate contains a target cleavage site reactive with said variant comprising amino acid residues P1, P2, P3 and P4 of said modified substrate where said P2 amino acid residue is histidine and said P1 residue is selected from the group consisting of tyrosine, phenylalanine, methionine, leucine and lysine.
 7. The process of claim 6 wherein said P1 residue is tyrosine.
 8. The process of claim 6 wherein said P1 residue is phenylalanine.
 9. The process of claim 6 wherein said P4 residue is selected from the group consisting of phenylalanine, isoleucine, methionine, alanine. leucine, lysine and valine.
 10. The process of claim 9 wherein said P4 residue is phenylalanine.
 11. The process of claim 2 wherein said modified substrate contains a target cleavage sequence reactive with said variant, said target cleavage sequence comprising the amino acid sequence phenylalanine-alanine-histidine-tyrosine.
 12. The process of claim 2 wherein said modified substrate contains a target cleavage sequence reactive with said variant, said target cleavage sequence comprising the amino acid sequence phenylalanine-alanine-histidine-phenylalanine.
 13. A process for cleaving a modified substrate containing histidine within a cleavage recognition sequence comprising contacting said modified substrate with a mutant subtilisin selected from the group consisting of H64A subtilisin, H64A:G166A subtilisin, H64A:E156S:G169A:Y217L subtilisin and H64A:E156S:G166A:G169A:Y217L subtilisin.
 14. The process of claim 13 wherein said mutant subtilisins further include the mutation S24C. 